Binding agent

ABSTRACT

A binding agent of the Formula A-a′:a-S-b:b′-B:X(n), wherein A as well as B is a monovalent binder, a′:a as well as b:b′ is a binding pair wherein a′ and a do not interfere with the binding of b to b′ and vice versa, S is a spacer of at least 1 nm in length, :X denotes a functional moiety bound either covalently or via a binding pair to at least one of a′, a, b, b′ or S, (n) is an integer and at least 1, - represents a covalent bond, and the linker a-S-b has a length of 6 to 100 nm. Also disclosed are methods of producing such binding agent and certain uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2011/073633, filed Dec. 21, 2011, which claims the benefit ofEuropean Patent Application No. 10196685.1, filed Dec. 23, 2010, thedisclosures of which are all hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 19, 2013, isnamed SEQUENCE_LISTING_(—)27205US.txt, and is seven thousand fivehundred and nineteen bytes in size.

BACKGROUND OF THE DISCLOSURE

Bispecific antibodies, or bispecific binding agents in general, areunique in the sense that they can bind simultaneously to two differentepitopes on one antigen or to two different antigens. This propertyenables the development of novel therapeutic and diagnostic strategiesthat are not possible with conventional monoclonal antibodies. A largepanel of bispecific dual binders, e.g. of bispecific antibody formatshas been developed and reflects the strong scientific as well ascommercial interest in these molecules.

Monoclonal antibodies (mAbs), being directed towards single epitopes onthe antigen, usually bind with affinities which are less than theavidity of polyclonal antisera. However, certain pairs of mAbs directedtowards different epitopes on the same antigen can bind that antigenmore effectively and with an avidity greater than the sum of theaffinities of the corresponding individual mAb alone.

However, the avidity constants for synergizing pairs of mAb or for achemically cross-linked bispecific F(ab′)2 is generally only up to 15times greater than the affinity constants for the individual mAb, whichis significantly less than the theoretical avidity expected for idealcombination between the reactants (Cheong, H. S., et al., Biochem.Biophys. Res. Commun. 173 (1990) 795-800). One reason for this might bethat the individual epitope/paratope interactions involved in asynergistic binding (resulting in a high avidity) must be orientated ina particular way relative to each other for optimal synergy.

BRIEF SUMMARY OF THE DISCLOSURE

The present disclosure relates to a binding agent of the FormulaA-a′:a-S-b:b′-B:X(n), wherein A as well as B is a monovalent binder,wherein a′:a as well as b:b′ is a binding pair wherein a′ and a do notinterfere with the binding of b to b′ and vice versa, wherein S is aspacer of at least 1 nm in length, wherein :X denotes a functionalmoiety bound either covalently or via a binding pair to at least one ofa′, a, b, b′ or S, wherein (n) is an integer and at least 1, wherein -represents a covalent bond, and wherein the linker a-S-b has a length of6 to 100 nm. Also disclosed are a method of producing such binding agentand certain uses thereof.

The present disclosure relates to a binding agent of the FormulaA-a′:a-S-b:b′-B:X(n), wherein A as well as B is a monovalent binder,wherein a′:a as well as b:b′ is a binding pair wherein a′ and a do notinterfere with the binding of b to b′ and vice versa, wherein S is aspacer of at least 1 nm in length, wherein :X denotes a functionalmoiety bound either covalently or via a binding pair to at least one ofa′, a, b, b′ or S, wherein (n) is an integer and at least 1, wherein -represents a covalent bond, and wherein the linker a-S-b has a length of6 to 100 nm.

Further disclosed is a method of making such binding agent and the useof such agent. e.g. in an immunoassay procedure.

The use of the novel binding agent, especially in an immunologicaldetection procedure is also described and claimed

BRIEF DESCRIPTION OF THE FIGURES

The features of this disclosure, and the manner of attaining them, willbecome more apparent and the disclosure itself will be better understoodby reference to the following description of embodiments of thedisclosure taken in conjunction with the accompanying drawing.

FIG. 1A is an analytical gel filtration experiments assessing efficiencyof the anti-pIGF1-R dual binder assembly. Diagrams A, B and C show theelution profile of the individual dual binder components(flourescein-ssFab′ 1.4.168, Cy5-ssFab′ 8.1.2 and linker DNA (T=0); Fab′denotes an Fab′-fragment conjugated to a single-strandedoligonucleotide). Diagram D shows the elution profile after the 3components needed to form the bi-valent binding agent had been mixed ina 1:1:1 molar ratio. The thicker (bottom) curve represents absorbancemeasured at 280 nm indicating the presence of the ssFab′ proteins or thelinker DNA, respectively. The thinner top curve in B) and D) (absorbanceat 495 nm) indicates the presence of fluorescein and the thinner topcurve in a) and the middle curve in d) (absorbance at 635 nm) indicatesthe presence of Cy5. Comparison of the elution volumes of the singledual binder components (VE_(ssFab′1.4.168)˜15 ml; VE_(ssFab′8.1.2)˜15ml; VE_(linker)˜16 ml) with the elution volume of the reaction mix(VE_(mix)˜12 ml) demonstrates that the dual binder assembly reaction wassuccessful (rate of yield: ˜90%). The major 280 nm peak that representsthe eluted dual binder nicely overlaps with the major peaks in the 495nm and 635 nm channel, proving the presence of both ssFab′ 8.1.2 andssFab′1.4.168 in the peak representing the bi-valent binding agent.

FIG. 1B is an analytical gel filtration experiments assessing efficiencyof the anti-pIGF1-R dual binder assembly. Diagrams A, B and C show theelution profile of the individual dual binder components(flourescein-ssFab′ 1.4.168, Cy5-ssFab′ 8.1.2 and linker DNA (T=0); Fab′denotes an Fab′-fragment conjugated to a single-strandedoligonucleotide). Diagram D shows the elution profile after the 3components needed to form the bi-valent binding agent had been mixed ina 1:1:1 molar ratio. The thicker (bottom) curve represents absorbancemeasured at 280 nm indicating the presence of the ssFab′ proteins or thelinker DNA, respectively. The thinner top curve in B) and D) (absorbanceat 495 nm) indicates the presence of fluorescein and the thinner topcurve in a) and the middle curve in d) (absorbance at 635 nm) indicatesthe presence of Cy5. Comparison of the elution volumes of the singledual binder components (VE_(ssFab′1.4.168)˜15 ml; VE_(ssFab′8.1.2)˜15ml; VE_(linker)˜16 ml) with the elution volume of the reaction mix(VE_(mix)˜12 ml) demonstrates that the dual binder assembly reaction wassuccessful (rate of yield: ˜90%). The major 280 nm peak that representsthe eluted dual binder nicely overlaps with the major peaks in the 495nm and 635 nm channel, proving the presence of both ssFab′ 8.1.2 andssFab′1.4.168 in the peak representing the bi-valent binding agent.

FIG. 1C is an analytical gel filtration experiments assessing efficiencyof the anti-pIGF1-R dual binder assembly. Diagrams A, B and C show theelution profile of the individual dual binder components(flourescein-ssFab′ 1.4.168, Cy5-ssFab′ 8.1.2 and linker DNA (T=0); Fab′denotes an Fab′-fragment conjugated to a single-strandedoligonucleotide). Diagram D shows the elution profile after the 3components needed to form the bi-valent binding agent had been mixed ina 1:1:1 molar ratio. The thicker (bottom) curve represents absorbancemeasured at 280 nm indicating the presence of the ssFab′ proteins or thelinker DNA, respectively. The thinner top curve in B) and D) (absorbanceat 495 nm) indicates the presence of fluorescein and the thinner topcurve in a) and the middle curve in d) (absorbance at 635 nm) indicatesthe presence of Cy5. Comparison of the elution volumes of the singledual binder components (VE_(ssFab′1.4.168)˜15 ml; VE_(ssFab′8.1.2)˜15ml; VE_(linker)˜16 ml) with the elution volume of the reaction mix(VE_(mix)˜12 ml) demonstrates that the dual binder assembly reaction wassuccessful (rate of yield: ˜90%). The major 280 nm peak that representsthe eluted dual binder nicely overlaps with the major peaks in the 495nm and 635 nm channel, proving the presence of both ssFab′ 8.1.2 andssFab′1.4.168 in the peak representing the bi-valent binding agent.

FIG. 1D is an analytical gel filtration experiments assessing efficiencyof the anti-pIGF1-R dual binder assembly. Diagrams A, B and C show theelution profile of the individual dual binder components(flourescein-ssFab′ 1.4.168, Cy5-ssFab′ 8.1.2 and linker DNA (T=0); Fab′denotes an Fab′-fragment conjugated to a single-strandedoligonucleotide). Diagram D shows the elution profile after the 3components needed to form the bi-valent binding agent had been mixed ina 1:1:1 molar ratio. The thicker (bottom) curve represents absorbancemeasured at 280 nm indicating the presence of the ssFab′ proteins or thelinker DNA, respectively. The thinner top curve in B) and D) (absorbanceat 495 nm) indicates the presence of fluorescein and the thinner topcurve in a) and the middle curve in d) (absorbance at 635 nm) indicatesthe presence of Cy5. Comparison of the elution volumes of the singledual binder components (VE_(ssFab′1.4.168)˜15 ml; VE_(ssFab′8.1.2)˜15ml; VE_(linker)˜16 ml) with the elution volume of the reaction mix(VE_(mix)˜12 ml) demonstrates that the dual binder assembly reaction wassuccessful (rate of yield: ˜90%). The major 280 nm peak that representsthe eluted dual binder nicely overlaps with the major peaks in the 495nm and 635 nm channel, proving the presence of both ssFab′ 8.1.2 andssFab′1.4.168 in the peak representing the bi-valent binding agent.

FIG. 2 presents a scheme of the Biacore™ experiment. Schematically andexemplarily, two binding molecules in solution are shown: The T0-T-Dig(linker 16), bi-valent binding agent and the T40-T-Dig (linker 15),bi-valent binding agent. Both these bi-valent binding agents only differin their linker-length (a central digoxigenylated T with no additional Tversus 40 additional Ts (20 on each side of the central T-Dig), betweenthe two hybridizing nucleic acid sequences). Furthermore, ssFab′fragments 8.1.2 and 1.4.168 were used.

FIG. 3 presents a Biacore™ sensorgram with overlay plot of threekinetics showing the interaction of 100 nM bi-valent binding agent(consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on theT40-T-Dig ssDNA-linker, i.e. linker 15) with the immobilized peptidepIGF-1R compared to the binding characteristics of 100 nM ssFab′ 1.4.168or 100 nM ssFab′ 8.1.2 to the same peptide. Highest binding performanceis obtained with the Dual Binder construct, clearly showing, that thecooperative binding effect of the Dual Binder increases affinity versusthe target peptide pIGF-1R.

FIG. 4 presents a Biacore™ sensorgram with overlay plot of threekinetics showing the interactions of the bi-valent binding agentconsisting of ssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on theT40-T-Dig ssDNA-linker, i.e. linker 15, with immobilized peptidespIGF-1R (phosphorylated IGF-1R), IGF-1R or pIR (phosphorylated insulinreceptor). Highest binding performance is obtained with the pIGF-1Rpeptide, clearly showing, that the cooperative binding effect of theDual Binder increases specificity versus the target peptide pIGF-1R ascompared to e.g. the phosphorylated insulin receptor peptide (pIR).

FIG. 5 presents a Biacore™ sensorgram with overlay plot of two kineticsshowing the interactions of 100 nM bi-valent binding agent consisting ofssFab′ 8.1.2 and ssFab′ 1.4.168 hybridized on the T40-T-DigssDNA-linker, i.e. linker 15, and a mixture of 100 nM ssFab′ 8.1.2 and100 nM ssFab′ 1.4.168 without linker DNA. Best binding performance isonly obtained with the bi-valent binding agent, whereas the mixture ofthe ssFab's without linker doesn't show an observable cooperativebinding effect, despite the fact that the total concentration of thesessFab's had been at 200 nM.

FIG. 6 presents a schematic drawing of a Biacore™ sandwich assay. Thisassay has been used to investigate the epitope accessibility for bothantibodies on the phosphorylated IGF-1R peptide. <MIgGFcy>R presents arabbit anti-mouse antibody used to capture the murine antibodyM-1.4.168. M-1.4.168 then is used to capture the pIGF-1R peptide.M-8.1.2 finally forms the sandwich consisting of M-1.4.168, the peptideand M-8.1.2

FIG. 7 presents a Biacore™ sensorgram showing the binding signal (thickline) of the secondary antibody 8.1.2. to the pIGF-1R peptide after thiswas captured by antibody 1.4.168 on the Biacore™ chip. The other signals(thin lines) are control signals: given are the lines from top to bottom500 nM 8.1.2, 500 nM 1.4.168; 500 nM target unrelated antibody<CKMM>M-33-IgG; and 500 nM target unrelated control antibody<TSH>M-1.20-IgG, respectively. No binding event could be detected in anyof these controls.

FIG. 8 presents a schematic drawing of the Biacore™ assay, presentingthe biotinylated dual binders on the sensor surface. On Flow Cell 1(=FC1) (not shown) amino-PEO-biotin was captured. On FC2, FC3 and FC4bi-valent binding agents with increasing linker length were immobilized.(shown are the dual binders on FC2 (T0-bi=only one central T-Bi) and FC4(T40-bi=one central T-Bi and 20 Ts each up- and downstream),respectively). Analyte 1: IGF-1R-peptide containing the M-1.4.168 ssFab′epitope at the right hand end of the peptide (top line)—the M-8.1.2ssFab′ phospho-epitope is not present, because this peptide is notphosphorylated; analyte 2: pIGF-1R peptide containing the M-8.1.2 ssFab′phospho-epitope (P) and the M-1.4.168 ssFab′ epitope (second line);analyte 3: pIR peptide, containing the cross reacting M-8.1.2 ssFab′phospho-epitope, but not the epitope for M-1.4.168 (third line).

FIG. 9 presents kinetic data of the Dual Binder experiment. T40-T-Bi(linker dual binder with ssFab′ 8.1.2 and ssFab′ 1.4.168 (=T40 in theFigure) shows a 1300-fold lower off-rate (kd=2.79E-05/s) versus pIGF-1Rwhen compared to pIR (kd=3.70E-02/s).

FIG. 10 presents a Biacore™ sensorgram, showing concentration dependentmeasurement of the T40-T-Bi dual binding agent vs. the pIGF-1R peptide(the phosphorylated IGF-1R peptide). The assay setup was as depicted inFIG. 8. A concentration series of the pIGF-1R peptide was injected at 30nM, 10 nM, 2×3.3 nM, 1.1 nM, 0.4 nM, 0 nM. The corresponding data aregiven in the table of FIG. 9.

FIG. 11 presents a Biacore™ sensorgram, showing concentration dependentmeasurement of the T40-T-Bi dual binding agent vs. the IGF-1R peptide(the non-phosphorylated IGF-1R peptide). The assay setup was as depictedin FIG. 8. A concentration series of the IGF-1R peptide was injected at300 nM, 100 nM, 2×33 nM, 11 nM, 4 nM, 0 nM. The corresponding data aregiven in the table of FIG. 9.

FIG. 12 presents a Biacore™ sensorgram, showing concentration dependentmeasurement of the T40-T-Bi dual binding agent vs. the pIR peptide (thephosphorylated insulin receptor peptide). The assay setup was asdepicted in FIG. 8. A concentration series of the pIR peptide wasinjected at 100 nM, 2×33 nM, 11 nM, 4 nM, 0 nM. The corresponding dataare given in the table depicted as FIG. 9.

Although the drawings represent embodiments of the present disclosure,the drawings are not necessarily to scale and certain features may beexaggerated in order to better illustrate and explain the presentdisclosure. The exemplifications set out herein illustrate an exemplaryembodiment of the disclosure, in one form, and such exemplifications arenot to be construed as limiting the scope of the disclosure in anymanner.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING 1. Antibody Fragments SEQ IDNO:1 V_(H) (mAb 1.4.168): QCDVKLVESG GGLVKPGGSL KLSCAASGFT FSDYPMSWVRQTPEKRLEWV ATITTGGTYT YYPDSIKGRF TISRDNAKNT LYLQMGSLQS EDAAMYYCTRVKTDLWWGLA YWGQGTLVTV SA SEQ ID NO:2 V_(L) (mAb 1.4.168): QLVLTQSSSASFSLGASAKL TCTLSSQHST YTIEWYQQQP LKPPKYVMEL KKDGSHTTGD GIPDRFSGSSSGADRYLSIS NIQPEDESIY ICGVGDTIKE QFVYVFGGGT KVTVLG SEQ ID NO:3 V_(H)(mAb 8.1.2): EVQLQQSGPA LVKPGASVKM SCKASGFTFT SYVIHWVKQK PGQGLEWIGYLNPYNDNTKY NEKFKGKATL TSDRSSSTVY MEFSSLTSED SAVYFCARRG IYAYDHYFDYWGQGTSLTVS S SEQ ID NO:4 V_(L) (mAb 8.1.2): QIVLTQSPAI MSASPGEKVTLTCSASSSVN YMYWYQQKPG SSPRLLIYDT SNLASGVPVR FSGSGSVTSY SLTISRMEAEDAATYYCQQW STYPLTFGAG TKLELK 2. Sequences of ssDNA

a) 17mer ssDNA (covalently bound with 5′ end to Fab′ of anti-TroponinTMAB a or Fab′ 1.4.168 to IGF-1R, respectively): 5′-AGT TCT ATC GTC GTCCA-3′(SEQ ID NO:5)b) 19mer ssDNA (covalently bound with 3′ end to Fab′ of anti-TroponinTMAB b or Fab′ 8.1.2 to phosphorylated IGF-1R, respectively): 5′-A GTCTAT TAA TGC TTC TGC-3′(SEQ ID NO:6)c) complementary 19mer ssDNA (used as part of a linker): 5′-G CAG AAGCAT TAA TAG ACT-3′(SEQ ID NO:7)d) complementary 17mer ssDNA (used as part of a linker): 5′-TGG ACG ACGATA GAA CT-3′(SEQ ID NO:8)

3. Sequences of Troponin T Epitopes

SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein U representsβ-Alanin. (The epitope “A” for antibody anti-Troponin antibody a.)SEQ ID NO:10=SLKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein O representsAmino-trioxa-octanoic-acid. (The epitope “B” for antibody anti-Troponinantibody b.)

4. Sequences of IGF-1R/IR Epitopes SEQ IDNO:11=FDERQPYAHMNGGRKNERALPLPQSST; IGF-1R (1340-1366)

SEQ ID NO:12=YEEHIPYTHMNGGKKNGRILTLPRSNPS; hIR(1355-1382)

5. Protein Linker and Tag-Sequences

SEQ ID NO:13=GGGGS (=G4S) motif (e.g. as part of a polypeptide linker)

SEQ ID NO:14=YPYDVPDYA (HA-Tag) SEQ ID NO:15=GLNDIFEAQKIEWHE (Avi-Tag)

Although the sequence listing represents an embodiment of the presentdisclosure, the sequence listing is not to be construed as limiting thescope of the disclosure in any manner and may be modified in any manneras consistent with the instant disclosure and as set forth herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

The embodiments disclosed herein are not intended to be exhaustive orlimit the disclosure to the precise form disclosed in the followingdetailed description. Rather, the embodiments are chosen and describedso that others skilled in the art may utilize their teachings.

The present disclosure relates to a binding agent of the Formula:A-a′:a-S-b:b′-B:X(n), wherein A as well as B is a monovalent binder,wherein a′:a as well as b:b′ is a binding pair wherein a′ and a do notinterfere with the binding of b to b′ and vice versa, wherein S is aspacer of at least 1 nm in length, wherein :X denotes a functionalmoiety bound either covalently or via a binding pair to at least one ofa′, a, b, b′ or S, wherein (n) is an integer and at least 1, wherein -represents a covalent bond, and wherein the linker a-S-b has a length of6 to 100 nm. As obvious the binding agent according to the presentdisclosure is a binding agent comprising at least two monovalent bindersof different specificity. In one embodiment the binding agent accordingto the present disclosure comprises two monovalent binders. In oneembodiment the binding agent according to the present disclosure is abi-valent or dual binding agent.

The generation of bispecific antibodies is e.g. described in WO2004/081051. In this application a bispecific antibody (BAb) comprisingtwo antibodies, each of which has a binding specificity to a differentepitope situated on the surface of a target structure are disclosed. Inorder to achieve the desired improvement in specificity two MAbs areused each having a relatively low binding affinity for its respectiveepitope. The BAbs produced provide high avidity for target tissue due tothe cumulative nature of the binding interactions but have much loweraffinity for cross-reactive non-target tissue due to the lower affinityof the individual MAbs used to produce them. Production of thesebispecific antibodies is quite complex and e.g. requires sophisticatedchemical coupling and purification steps.

Bispecific monoclonal antibodies also represent quite interesting noveltherapeutic modalities. A broad spectrum of bispecific antibody formatshas been designed and developed (see e.g. Fischer, N. and Leger, O.,Pathobiology 74 (2007) 3-14). Such bispecific therapeutic monoclonalscan e.g. be obtained by chemical cross-linking, interaction ofappropriately engineered protein domains, completely recombinant, etc.Obviously, recombinant engineering of each of the binders and carefulpurification of the desired heterodimer from biochemically alikehomo-dimers represent some of the challenges encountered.

Chelating recombinant antibodies (CRAbs), originally described by Neri,D. et al. (1995) represent a species of very high affinity antibodies,where two scFvs specific for non-overlapping epitopes on the sameantigen molecule are connected by a flexible linker polypeptide. Theoriginal modeled and designed anti-hen egg lysozyme (HEL) CRAB employedan 18 amino acid linker polypeptide to span the distance between the twoscFv antibodies and the resulting affinity enhancement was subsequentlyshown to be up to 100-fold higher than the superior of the two scFvs asshown by a variety of biophysical methods (Neri, D. et al., J. Mol.Biol. 246 (1995) 367-373).

Wright M. J. and Deonarain M. P., (Molecular Immunology 44 (2007)2860-2869) developed a phage display library for generation of chelatingrecombinant antibodies. The library described there uses expressionvectors construed in such way to provide for dual binders having linkerpeptides of various length in between the two binding entities.Selection of the best binder, i.e. the dual binder with the optimallength of such linker, is thereby facilitated. However, for each suchchelating recombinant antibody a full library of recombinant expressionsystems (allowing for expression of a “binder 1-linker (of variablelength)-binder 2” polypeptide) has to be construed.

As outlined above in a cursory manner, the manufacturing of bispecificdual binders remains quite challenging and requires sophisticatedtechniques to identify, construe and produce individually each of thosebispecific binding agents. The frequent need for derivatizing, e.g.,labeling such a bispecific binding agent even adds a further level ofcomplexity.

The instant disclosure provides the surprising disclosure and findingsthat at least some of the disadvantages known from the prior art can beovercome by way of the novel bispecific binding agents and methodsdisclosed in the present embodiment.

As the skilled artisan will appreciate the binding agent described inthe present disclosure can be isolated and purified as desired. In oneembodiment the present disclosure relates to an isolated binding agentas disclosed herein. An “isolated” binding agent is one which has beenidentified and separated and/or recovered from e.g. the reagent mixtureused in the synthesis of such binding agent. Unwanted components of suchreaction mixture are e.g. monovalent binders that did not end up in thedesired binding agent. In one embodiments, the binding agent is purifiedto greater than 80%. In some embodiments, the binding agent is purifiedto greater than 90%, 95%, 98% or 99% by weight, respectively. In caseboth monovalent binders of the binding agent according to the presentdisclosure are polypeptides purity is e.g. easily determined by SDS-PAGEunder reducing or nonreducing conditions using, for example, Coomassieblue or silver stain in protein detection. In case purity is assessed onthe nucleic acid level, size chromatography is applied to separate thebinding agent from side products and the OD at 260 nm is monitored toassess its purity.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an antibody” means one antibody or morethan one antibody.

The terms “polypeptide” and “protein” are used inter-changeably. Apolypeptide in the sense of the present disclosure consists of at least5 amino acids linked by alpha amino peptidic bonds.

A “target molecule” is a biomolecule of interest for which a method fordetermination or measurement is sought. Exemplary target molecules arelipoproteins, polypeptides, complexes of polypeptides, secondarilymodified polypeptides and complexes between polypeptides and nucleicacids. In one embodiment a target molecule is a polypeptide.

A “monovalent binder” (A and B, respectively, in Formula I) according tothe present disclosure is a molecule interacting with a target molecule,e.g. with a target polypeptide at a single site (i.e. the specificbinding site). In case monovalent antibodies or antibody fragments areused as a binder this site is called the paratop.

As will be appreciated, the monovalent binders A and B, respectively,each specifically bind their corresponding antigen. In an exemplaryembodiment the epitopes specifically bound by the monovalent binders Aand B do not overlap. As the skilled artisan will appreciate the termspecific is used to indicate that other biomolecules present in thesample do not significantly bind to the binding agent used. In someembodiments, for a specific binder the level of binding affinity to abiomolecule other than the target molecule results in a binding affinitywhich is only 10% or less, for example, in some embodiments only 5% orless of the affinity it has to the specifically bound target molecule.

Examples of monovalent binders are peptides, peptide mimetics, aptamers,spiegelmers, darpins, ankyrin repeat proteins, Kunitz type domains,single domain antibodies (see: Hey, T. and Fiedler, E., et al., TrendsBiotechnol. 23 (2005) 514-522), and monovalent fragments of antibodies.

In certain embodiments the monovalent binder is a polypeptide. Inexemplary embodiments each of the monovalent binders A and B,respectively is a polypeptide.

In certain embodiments the monovalent binder A and B, respectively, is amonovalent antibody fragment, for example a monovalent fragment derivedfrom a monoclonal antibody.

Monovalent antibody fragments include, but are not limited to Fab,Fab′-SH, single domain antibody, Fv, and scFv fragments, as providedbelow.

In exemplary embodiments at least one of the monovalent binders is asingle domain antibody, an Fab-fragment or an Fab′-fragment of amonoclonal antibody.

It also represents an exemplary embodiment that in the binding agentdisclosed herein both the monovalent binders are derived from monoclonalantibodies and are Fab-fragments, or Fab′-fragments or an Fab-fragmentand an Fab′-fragment. Also, some embodiments include the binding agentcomprising two Fab-fragments as the monovalent binders A and B.

Monoclonal antibody techniques allow for the production of extremelyspecific binding agents in the form of specific monoclonal antibodies orfragments thereof. Particularly well known in the art are techniques forcreating monoclonal antibodies, or fragments thereof, by immunizingmice, rabbits, hamsters, or any other mammal with a polypeptide ofinterest. Another method of creating monoclonal antibodies, or fragmentsthereof, is the use of phage libraries of sFv (single chain variableregion), specifically human sFv. (See e.g., Griffiths et al., U.S. Pat.No. 5,885,793; McCafferty et al., WO 92/01047; Liming et al., WO99/06587).

Antibody fragments may be generated by traditional means, such asenzymatic digestion or by recombinant techniques. For a review ofcertain antibody fragments, see Hudson, P. J. et al., Nat. Med. 9 (2003)129-134.

An Fv is a minimum antibody fragment that contains a completeantigen-binding site and is devoid of constant region. In oneembodiment, a two-chain Fv species consists of a dimer of one heavy- andone light-chain variable domain in tight, non-covalent association. Inone embodiment of a single-chain Fv (scFv) species, one heavy- and onelight-chain variable domain can be covalently linked by a flexiblepeptide linker such that the light and heavy chains can associate in adimeric structure analogous to that in a two-chain Fv species. For areview of scFv, see, e.g., Plueckthun, In: The Pharmacology ofMonoclonal Antibodies, Vol. 113, Rosenburg and Moore (eds.),Springer-Verlag, New York (1994), pp. 269-315; see also WO 93/16185; andU.S. Pat. Nos. 5,571,894 and 5,587,458. Generally, six hyper variableregions (HVRs) confer antigen-binding specificity to an antibody.However, even a single variable domain (or half of an Fv comprising onlythree HVRs specific for an antigen) has the ability to recognize andbind antigen.

An Fab fragment contains the heavy- and light-chain variable domains andalso contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup.

Various techniques have been developed for the production of antibodyfragments. Traditionally, antibody fragments were derived viaproteolytic digestion of intact antibodies (see, e.g., Morimoto, K. etal., Journal of Biochemical and Biophysical Methods 24 (1992) 107-117;and Brennan, M. et al., Science 229 (1985) 81-83). For example, papaindigestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily.

Antibody fragments can also be produced directly by recombinant hostcells. Fab, Fv and scFv antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of these fragments. Antibody fragments can be isolated from theantibody phage libraries according to standard procedures.Alternatively, Fab′-SH fragments can be directly recovered from E. coli.(Carter, P. et al., Bio/Technology 10 (1992) 163-167). Mammalian cellsystems can be also used to express and, if desired, secrete antibodyfragments.

In certain embodiments, a monovalent binder of the present disclosure isa single-domain antibody. A single-domain antibody is a singlepolypeptide chain comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,U.S. Pat. No. 6,248,516 B1). In one embodiment, a single-domain antibodyconsists of all or a portion of the heavy chain variable domain of anantibody.

The term “oligonucleotide” or “nucleic acid sequence” as used herein,generally refers to short, generally single stranded, polynucleotidesthat comprise at least 8 nucleotides and at most about 1000 nucleotides.In an exemplary embodiment an oligonucleotide will have a length of atleast 9, 10, 11, 12, 15, 18, 21, 24, 27 or 30 nucleotides. In anexemplary embodiment an oligonucleotide will have a length of no morethan 200, 150, 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides.The description given below for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term oligonucleotide is to be understood broadly and includes DNAand RNA as well as analogs and modification thereof.

An oligonucleotide may for example contain a substituted nucleotidecarrying a substituent at the standard bases deoxyadenosine (dA),deoxyguanosine (dG), deoxycytosine (dC), deoxythymidine (dT),deoxyuracil (dU). Examples of such substituted nucleobases are:5-substituted pyrimidines like 5 methyl dC, aminoallyl dU or dC,5-(aminoethyl-3-acrylimido)-dU, 5 propinyl dU or dC, 5 halogenated-dU ordC; N substituted pyrimidines like N4 ethyl dC; N substituted purineslike N6 ethyl dA, N2 ethyl dG; 8 substituted purines like8-[6-amino)-hex-1-yl]-8-amino-dG or dA, 8 halogenated dA or dG, 8-alkyldG or dA; and 2 substituted dA like 2 amino dA.

An oligonucleotide may for example contain a substituted nucleotidecarrying a substituent at the standard bases deoxyadenosine (dA),deoxyguanosine (dG), deoxycytosine (dC), deoxythymidine (dT),deoxyuracil (dU). Examples of such substituted nucleobases are:5-substituted pyrimidines like 5 methyl dC, aminoallyl dU or dC,5-(aminoethyl-3-acrylimido)-dU, 5-propinyl-dU or -dC, 5 halogenated-dUor -dC; N substituted pyrimidines like N4-ethyl-dC; N substitutedpurines like N6-ethyl-dA, N2-ethyl-dG; 8 substituted purines like8-[6-amino)-hex-1-yl]-8-amino-dG or -dA, 8 halogenated dA or dG, 8-alkyldG or dA; and 2 substituted dA like 2 amino dA.

An oligonucleotide may contain a nucleotide or a nucleoside analog. I.e.the naturally occurring nucleobases can be exchanged by using nucleobaseanalogs like 5-Nitroindol d riboside; 3 nitro pyrrole d riboside,deoxyinosine (dI), deoyxanthosine (dX); 7 deaza-dG, -dA, -dI or -dX;7-deaza-8-aza-dG, -dA, -dI or -dX; 8-aza-dA, -dG, -dI or -dX; dFormycin; pseudo dU; pseudo iso dC; 4 thio dT; 6 thio dG; 2 thio dT; isodG; 5-methyl-iso-dC; N8-linked 8-aza-7-deaza-dA; 5,6-dihydro-5-aza-dC;and etheno-dA or pyrollo-dC. As obvious to the skilled artisan, thenucleobase in the complementary strand has to be selected in such mannerthat duplex formation is specific. If, for example, 5-methyl-iso-dC isused in one strand (e.g. (a)) iso dG has to be in the complementarystrand (e.g. (a′)).

The oligonucleotide backbone may be modified to contain substitutedsugar residues, sugar analogs, modifications in the internucleosidephosphate moiety, and/or be a PNA.

An oligonucleotide may for example contain a nucleotide with asubstituted deoxy ribose like 2′-methoxy, 2′-fluoro, 2′-methylseleno,2′-allyloxy, 4′-methyl dN (wherein N is a nucleobase, e.g., A, G, C, Tor U).

Sugar analogs are for example Xylose; 2′,4′ bridged Ribose like (2′-O,4′-C methylene)- (oligomer known as LNA) or (2′-O, 4′-C ethylene)-(oligomer known as ENA); L-ribose, L-d-ribose, hexitol (oligomer knownas HNA); cyclohexenyl (oligomer known as CeNA); altritol (oligomer knownas ANA); a tricyclic ribose analog where C3′ and C5′ atoms are connectedby an ethylene bridge that is fused to a cyclopropane ring (oligomerknown as tricycloDNA); glycerin (oligomer known as GNA); Glucopyranose(oligomer known as Homo DNA); carbaribose (with a cyclopentan instead ofa tetrahydrofuran subunit); hydroxymethyl-morpholin (oligomers known asmorpholino DNA).

A great number of modification of the internucleosidic phosphate moietyare also known not to interfere with hybridization properties and suchbackbone modifications can also be combined with substituted nucleotidesor nucleotide analogs. Examples are phosphorthioate, phosphordithioate,phosphoramidate and methylphosphonate oligonucleotides.

PNA (having a backbone without phosphate and d-ribose) can also be usedas a DNA analog.

The above mentioned modified nucleotides, nucleotide analogs as well asoligonucleotide backbone modifications can be combined as desired in anoligonucleotide in the sense of the present disclosure.

The linker L consisting of a-S-b has a length of 6 to 100 nm. In someembodiments the linker L consisting of a-S-b has a length of 6 to 80 nm.Also, in some cases the linker has a length of 6 to 50 nm or of 6 to 40nm. In some embodiments the linker will have a length of 10 nm or longeror of 15 nm in length or longer. In some embodiments the linker hasbetween 10 nm and 50 nm in length. In some embodiments a and b,respectively, are binding pair members and have a length of at least 2.5nm each.

The length of non-nucleosidic entities of a given linker (a-S-b) intheory and by complex methods can be calculated by using known bonddistances and bond angles of compounds which are chemically similar tothe non-nucleosidic entities. Such bond distances are summarized forsome molecules in standard text books: CRC Handbook of Chemistry andPhysics, 91st edition, 2010-2011, section 9. However, exact bonddistances vary for each compound. There is also variability in the bondangles.

It is therefore more practical to use an average parameter (an easy tounderstand approximation) in such calculation.

In the calculation of a spacer or a linker length the followingapproximations apply: a) for calculating lengths of nonnucleosidicentities an average bond length of 130 pm with an bond angle of 180°independently of the nature of the linked atoms is used; b) onenucleotide in a single strand is calculated with 500 pm and c) onenucleotide in a double strand is calculated with 330 pm.

The value of 130 pm is based on calculation of the distance of the twoterminal carbonatoms of a C(sp3)-C(sp3)-C(sp3) chain with a bond angleof 109° 28′ and a distance of 153 pm between two C(sp3) which is approx250 pm which translates with an assumed bond angle of 180° to and bonddistance between two C(Sp3) with 125 pm. Taking in account thatheteroatoms like P and S and sp2 and sp1 C atoms could also be part ofthe spacer the value 130 pm is taken. If a spacer comprises a cyclicstructure like cycloalkyl or aryl the distance is calculated inanalogous manner, by counting the number of the bonds of said cyclicstructure which are part of the overall chain of atoms that are definingthe distance.

The spacer S can be construed as required to e.g. provide for thedesired length as well as for other desired properties. The spacer cane.g. be fully or partially composed of naturally occurring ornon-naturally occurring amino acids, of phosphate-sugar units e.g. a DNAlike backbone without nucleobases, of glyco-peptidic structures, or atleast partially of saccharide units or at least partially ofpolymerizable subunits like glycols or acryl amide.

The length of spacer S in a binding agent according to the presentdisclosure may be varied as desired. In order to easily make availablespacers of variable length, a library, some embodiments may have asimple synthetic access to the spacers of such library. A combinatorialsolid phase synthesis of a spacer may be present in some embodiments.Since spacers have to synthesized up to a length of about 100 nm, thesynthesis strategy is chosen in such a manner that the monomericsynthetic building blocks are assembled during solid phase synthesiswith high efficiency. The synthesis of deoxy oligonucleotides based onthe assembly of phosphoramidite as monomeric building blocks perfectlymeet this requirements. In such spacer monomeric units within a spacerare linked in each case via a phosphate or phosphate analog moiety.

The spacer S can contain free positively or/and negatively chargedgroups of polyfunctional amino-carboxylic acids, e.g. amino, carboxylateor phosphate. For example the charge carriers can be derived fromtrifunctional aminocarboxylic acids which contain a) an amino group andtwo carboxylate groups or b) two amino groups and one carboxylate group.Examples of such trifunctional aminocarboxylic acids are lysine,ornithine, hydroxylysine, α,β-diamino propionic acid, arginine, asparticacid and glutamic acid, carboxy glutamic acid and symmetrictrifunctional carboxylic acids like those described in EP-A-0 618 192 orU.S. Pat. No. 5,519,142. Alternatively one of the carboxylate groups inthe trifunctional aminocarboxylic acids a) can be replaced by aphosphate, sulphonate or sulphate group. An example of such atrifunctional amino acid is phosphoserine.

The spacer S can also contain uncharged hydrophilic groups. Examples ofuncharged hydrophilic groups include ethylene oxide or polyethyleneoxide groups, for example, with at least three ethylene oxide units,sulphoxide, sulphone, carboxylic acid amide, carboxylic acid ester,phosphonic acid amide, phosphonic acid ester, phosphoric acid amide,phosphoric acid ester, sulphonic acid amide, sulphonic acid ester,sulphuric acid amide and sulphuric acid ester groups. The amide groupsmay be primary amide groups, for example carboxylic acid amide residuesin amino acid side groups e.g. the amino acids asparagine and glutamine.The esters may also be derived from hydrophilic alcohols, in particularC1-C3 alcohols or diols or triols.

In one embodiment the spacer S is composed of one type of monomer. Forexample, the spacer is composed exclusively of amino acids, of sugarresidues, of diols, of phospho-sugar units or it can be a nucleic acid,respectively.

In one embodiment, the spacer is DNA. In an exemplary embodiment thespacer is the L-stereoisomer of DNA also known as beta-L-DNA, L-DNA ormirror image DNA. L-DNA features advantages like orthogonalhybridization behaviour, which means that a duplex is formed onlybetween two complementary single strands of L-DNA but no duplex isformed between a single strand of L-DNA and the complementary DNAstrand, nuclease resistance and ease of synthesis even of a long spacer.As pointed out ease of synthesis and variability in spacer length areimportant for a spacer library. Spacers of variable length are extremelyutile in identifying the binding agent according to the presentdisclosure having a spacer of optimal length thus providing for theoptimal distance between the two monovalent binders.

Spacer building blocks, as the name says, can be used to introduce aspacing moiety into the spacer S or to build the spacer S of the linkera-S-b.

Different numbers and kinds of non-nucleotidic as well nucleotidicspacer building blocks are at hand for introducing spacing moieties.

Many different non nucleotidic bifunctional spacer building blocks areknown in literature and a great variety is commercially available. Thechoice of the non nucleotidic bifunctional spacer building isinfluencing the charge and flexibility of the spacer molecule.

In bifunctional spacer building blocks a hydroxyl group which isprotected with an acid labile protecting group is connected to aphosphoramidite group.

Bifunctional spacer building blocks in one embodiment arenon-nucleosidic compounds. For example, such spacers are C2-C18 alkyl,alkenyl, alkinyl carbon chains, whereas said alkyl, alkenyl, alkinylchains may be interrupted by additional ethyleneoxy and/or amidemoieties or quarternized cationic amine moieties in order to increasehydrophilicity of the linker. Cyclic moieties like C5-C6-cycloalkyl,C4N, C5N, C4O, C5O-heterocycloalkyl, phenyl which are optionallysubstituted with one or two C1-C6 alkyl groups can also be used asnonnucleosidic bifunctional spacer moieties. Exemplary bifunctionalbuilding blocks comprise C3-C6 alkyl moieties and tri- tohexa-ethyleneglycol chains. Table I shows some examples of nucleotidicbifunctional spacer building blocks with different hydrophilicity,different rigidity and different charges. One oxygen atom is connectedto an acid labile protecting group such as dimethoxytrityl and the otheris part of a phosphoramidite.

TABLE I Examples of non-nucleotidic bifunctional spacer building blocksNon-nucleotidic bifunctional spacer building blocks Reference

Seela, F., Nucleic Acids Research 15 (1987) 3113-3129

Iyer, R.P., Nucleic Acids Research 18 (1990) 2855-2859

WO 89/02931 A1

EP 1 538 221

US 2004/224372

WO 2007/069092

A simple way to build the spacer S or to introduce spacing moieties intothe spacer S is to use standard D or L nucleoside phosphoramiditebuilding blocks. In one embodiment a single strand stretch of dT isused. This is advantageous, because dT does not carry a base protectinggroup.

Hybridization can be used in order to vary the spacer length (distancebetween the binding pair members a and b) and the flexibility of thespacer, because the double strand length is reduced compared to thesingle strand and the double strand is more rigid than a single strand.

For hybridization in one embodiment oligonucleotides modified with afunctional moiety X are used. The oligonucleotide used for hybridizationcan have one or two terminal extensions not hybridizing with the spacerand/or is branched internally.

Such terminal extensions that are not hybridizing with the spacer (andnot interfering with the binding pairs a:a′ and b:b′) can be used forfurther hybridization events. In one embodiment an oligonucleotidehybridizing with a terminal extension is a labeled oligonucleotide. Thislabeled oligonucleotide again may comprise terminal extensions or beingbranched in order to allow for further hybridization, thereby apolynucleotide aggregate or dendrimer can be obtained. Apoly-oligonucleic acid dendrimer may be used in order to produce apolylabel. or in order to get a high local concentration of X.

In one embodiment the spacer S has a backbone length of 1 to 100 nm.With other words here the groups a and b of Formula I are between 1 and100 nm apart. In one embodiment a and b, respectively, each are abinding pair member and the spacer S has a backbone length of 1 to 95nm.

“a′:a” as well as “b:b′” each independently represent a binding pair. Inone embodiment each of the binding pair members a and b, respectively,has a length of at least 2.5 nm.

a and a′ are the members of the binding pair a′:a and b and b′ are themembers of the binding pair b:b′, respectively. Each member of a bindingpair may be of a molecular weight of 10 kD or below, for example. Infurther embodiments the molecular weight of each binder of such bindingpair is 8, 7, 6, 5 or 4 kD or below.

The binding affinity for (within) a binding pair, a:a′ or b′:b,respectively, is at least 10⁸ l/mol. Both binding pairs are different.For a binding pair difference is e.g. acknowledged if the affinity forthe reciprocal binding, e.g. binding of a as well as a′ to b or b′ is10% of the affinity within the pair a:a′ or lower. Also, the reciprocalbinding, i.e. binding of a as well as a′ to b or b′, respectively, maybe 5% of the affinity within the pair a:a′ or lower, or if it is 2% ofthe affinity within the pair a:a′ or lower. In one embodiment thedifference is so pronounced that the reciprocal (cross-reactive) bindingis 1% or less as compared to the specific binding affinity within abinding pair.

In one embodiment a′:a and b:b′ are binding pairs and the members of thebinding pairs a′:a and b:b′ are selected from the group consisting ofleucine zipper domain dimers and hybridizing nucleic acid sequences. Inone embodiment both binding pairs represent leucine zipper domaindimers. In one embodiment both binding pairs are hybridizing nucleicacid sequences.

The term “leucine zipper domain” is used to denote a commonly recognizeddimerization domain characterized by the presence of a leucine residueat every seventh residue in a stretch of approximately 35 residues.Leucine zipper domains are peptides that promote oligomerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz, H. W. et al.,Science 240 (1988) 1759-1764), and have since been found in a variety ofdifferent proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.Examples of leucine zipper domains suitable for producing solublemultimeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe, H. J. et al., FEBS Lett. 344 (1994) 191-195.

Leucine zipper domains form dimers (binding pairs) held together by analpha-helical coiled coil. A coiled coil has 3.5 residues per turn,which means that every seventh residue occupies an equivalent positionwith respect to the helix axis. The regular array of leucines inside thecoiled coil stabilizes the structure by hydrophobic and Van der Waalsinteractions.

If leucine zipper domains form the first binding pair (a′:a) and thesecond binding pair (b:b′), both leucine zipper sequences are different,i.e. sequences a and a′ do not bind to b and b′. Leucine zipper domainsmay be isolated from natural proteins known to contain such domains,such as transcription factors. One leucine zipper domain may e.g. comefrom the transcription factor fos and a second one from thetranscription factor jun. Leucine zipper domains may also be designedand synthesized artificially, using standard techniques for synthesisand design known in the art.

In an exemplary embodiment both members of the binding pairs a′:a andb:b′, i.e. a, a′, b and b′ represent leucine zipper domains and thespacer S consists of amino acids. In this embodiment production of theconstruct a-S-b is easily possible. Varying the length of such spacer Sas desired is straightforward for a person skilled in the art. Suchpolypeptide can be synthesized or recombinantly produced.

E.g., recombinant fusion proteins comprising a spacer polypeptide fusedto a leucine zipper peptide at the N-terminus and to a leucine zipperpeptide at the C-terminus can be expressed in suitable host cellsaccording to standard techniques. A DNA sequence coding for a desiredpeptide spacer can be inserted between a sequence coding for a member ofa first leucine zipper domain a and in the same reading frame a DNAsequence coding for a member of a second leucine zipper domain b.

The spacer S, if the linker a-S-b is a polypeptide in one embodimentcomprises once or several times a GGGGS (SEQ ID NO:13) amino acidsequence motif. The spacer S may also comprise a tag sequence. The tagsequence may be selected from commonly used protein recognition tagssuch as YPYDVPDYA (HA-Tag) (SEQ ID NO:14) or GLNDIFEAQKIEWHE (Avi-Tag)(SEQ ID NO:15).

In an exemplary embodiment both binding pairs (a′:a) and (b:b′) arehybridizing nucleic acid sequences.

As indicated already by nomenclature, a and a′ as well as b and b′hybridize to one another, respectively. The nucleic acid sequencescomprised in a and a′ one the one hand and in b and b′ on the other handare different. With other words the sequences of in the binding paira′:a do not bind to the sequences of the binding pair b:b′,respectively, and vice versa. In one embodiment the present disclosurerelates to an at least dual binding agent of Formula I, wherein thebinding pairs a:a′ and b:b′, respectively, both are hybridizing nucleicacid sequences and wherein the hybridizing nucleic acid sequences of thedifferent binding pairs a′:a and b:b′ do not hybridize with one another.With other words a and a′ hybridize to each other but do not bind to anyof b or b′ or interfere with their hybridization and vice versa.Hybridization kinetics and hybridization specificity can easily bemonitored by melting point analyses. Specific hybridization of a bindingpair (e.g. a:a′) and non-interference (e.g. with b or b′) isacknowledged, if the melting temperature for the pair a:a′ as comparedto any possible combination with b or b′, respectively, (i.e. a:b; a:b′;a′:b and a′:b′) is at least 20° C. higher.

The nucleic acid sequences forming a binding pair, e.g. (a:a′) or anyother nucleic acid sequence-based binding pair, may compromise anynaturally occurring nucleobase or an analogue thereto and may have amodified or an un-modified backbone as described above, provided it iscapable of forming a stable duplex via multiple base pairing. Stablemeans that the melting temperature of the duplex is higher than 37° C.In some cases, the double strand consists of two fully complementarysingle strands. However mismatches or insertions are possible as long asthe a stability at 37° C. is given.

As the skilled artisan will appreciate a nucleic acid duplex can befurther stabilized by interstrand crosslinking. Several appropriatecross-linking methods are known to the skilled artisan, e.g. methodsusing psoralen or based on thionucleosides.

The nucleic acid sequences representing the members of a binding pairmay consist of between 12 and 50 nucleotides. Also, in some embodimentssuch nucleic acid sequences will consist of between 15 and 35nucleotides.

RNAses are ubiquitous and special care has to be taken to avoid unwanteddigestion of RNA-based binding pairs and/or spacer sequences. While itcertainly is possible to use, e.g. RNA-based binding pairs and/orspacers, binding pairs and/or spacers based on DNA represent anexemplary embodiment.

Appropriate hybridizing nucleic acid sequences can easily be designed toprovide for more than two pairs of orthogonal complementaryoligonucleotides, allowing for an easy generation and use of more thantwo binding pairs. Another advantage of using hybridizing nucleic acidsequences in a binding agent of the present disclosure is thatmodifications can be easily introduced into a nucleic acid sequences.Modified building blocks are commercially available which e.g. allow foran easy synthesis of a linker comprising a functional moiety. Suchfunctional moiety can be easily introduced at any desired position andin any of the structures a and a′ as well as b and b′ and/or S, providedthey represent an oligonucleotide.

In some embodiments the spacer S comprised in a binding agent accordingto Formula I is a nucleic acid. In some embodiments both binding pairsare hybridizing nucleic acid sequences and the spacer S also is anucleic acid. In this embodiment the linker L consisting of a-S-b is anoligonucleotide.

In case the spacer S as well as the sequences a, a′, b and b′ all areoligonucleotide sequences it is easily possible to provide for andsynthesize a single oligonucleotide representing the linker L comprisingS and the members a and b of the binding pairs a′:a and b:b′,respectively. In case the monovalent binders A and B, respectively, arepolypeptides, they can each be coupled easily to the hybridizing nucleicacid sequences a′ and b′, respectively. The length of the spacer Scomprised in such construct can easily be varied in any desired manner.Based on the three constructs a-S-b, A-a′ and b′-B the binding agent ofFormula I can be most easily obtained according to standard proceduresby hybridization between a′:a and b:b′, respectively. When spacers ofdifferent length are used, the resulting constructs, provide forotherwise identical binding agents, yet having a different distance inbetween the monovalent binders A and B. This allows for optimal distanceand/or flexibility.

In some embodiments the spacer S as well as the sequences a, a′, b andb′ are DNA.

The enantiomeric L-DNA, is known for its orthogonal hybridizationbehavior, its nuclease resistance and for ease of synthesis ofoligonucleotides of variable length. This ease of variability in linkerlength via designing appropriate spacers is important for optimizing thebinding of a binding agent as disclosed herein to its antigen orantigens.

In some embodiments the linker L (=a-S-b) is enantiomeric L-DNA orL-RNA. In an exemplary embodiment linker a-S-b is enantiomeric L-DNA. Inan exemplary embodiment a, a′, b and b′ as well as the spacer S areenantiomeric L-DNA or L-RNA. In an exemplary embodiment a, a′, b and b′as well as the spacer S are enantiomeric L-DNA.

In one embodiment the spacer S is an oligonucleotide and is synthesizedin two portions comprising ends hybridizable with each other. In thiscase the spacer S can be simply constructed by hybridization of thesehybridizable ends with one another. The resulting spacer constructcomprises an oligonucleotide duplex portion. As obvious, in case thespacer is construed that way, the sequence of the hybridizableoligonucleotide entity forming said duplex is chosen in such a mannerthat no hybridization or interference with the binding pairs a:a′ andb:b′ can occur.

As already described above the monovalent specific binders A and B ofFormula I may be nucleic acids. In one embodiment of the presentdisclosure a′, a, b, b′, A, B and s all are oligonucleotide sequences.In this embodiment the sub-units A-a′, a-S-b and b′-B of Formula I caneasily and independently be synthesized according to standard proceduresand combined by hybridization according to convenient standardprocedures. The functional moiety X may be selected from the groupconsisting of a binding group, a labeling group, an effector group and areactive group.

If more than one functional moiety X is present, each such functionalmoiety can in each case be independently a binding group, a labelinggroup, an effector group or a reactive group.

In one embodiment the functional moiety X may be selected from the groupconsisting of a binding group, a labeling group and an effector group.

In one embodiment the group X is a binding group. As obvious to a personskilled in the art, the binding group X will be selected to have nointerference with the pairs a′:a and b:b′.

Examples of binding groups are the partners of a bioaffine binding pairwhich can specifically interact with the other partner of the bioaffinebinding pair. Suitable bioaffine binding pairs are hapten or antigen andantibody; biotin or biotin analogues such as aminobiotin, iminobiotin ordesthiobiotin and avidin or streptavidin; sugar and lectin,oligonucleotide and complementary oligonucleotide, receptor and ligand,e.g., steroid hormone receptor and steroid hormone. In one embodiment Xis a binding group and is covalently bound to at least one of a′, a, b,b′ or S of the compound of Formula I. According to some embodiments thesmaller partner of a bioaffine binding pair, e.g. biotin or an analoguethereto, a receptor ligand, a hapten or an oligonucleotide is covalentlybound to at least one of a′, a, S, b or b′ as defined above.

In one embodiment functional moiety X is a binding group selected fromhapten; biotin or biotin analogues such as aminobiotin, iminobiotin ordesthiobiotin; oligonucleotide and steroid hormone.

In one embodiment the functional moiety X is a reactive group. Thereactive group can be selected from any known reactive group, likeAmino, Sulfhydryl, Carboxylate, Hydroxyl, Azido, Alkinyl or Alkenyl. Inone embodiment the reavtive group is selected from Maleinimido,Succinimidyl, Dithiopyridyl, Nitrophenylester, Hexafluorophenylester.

In one embodiment the functional moiety X is a labeling group. Thelabeling group can be selected from any known detectable group. Theskilled artisan will choose the number of labels as appropriate for bestsensitivity with least quenching.

The labeling group can be selected from any known detectable group. Inone embodiment the labeling group is selected from dyes like luminescentlabeling groups such as chemiluminescent groups e.g. acridinium estersor dioxetanes or fluorescent dyes e.g. fluorescein, coumarin, rhodamine,oxazine, resorufin, cyanine and derivatives thereof, luminescent metalcomplexes such as ruthenium or europium complexes, enzymes as used forCEDIA (Cloned Enzyme Donor Immunoassay, e.g. EP 0 061 888),microparticles or nanoparticles e.g. latex particles or metal sols, andradioisotopes.

In one embodiment the labeling group is a luminescent metal complex andthe compound has a structure of the general formula (II):

[M(L₁L₂L₃)]_(n)-Y-X_(m)A  (II)

in which M is a divalent or trivalent metal cation selected from rareearth or transition metal ions, L₁, L₂ and L₃ are the same or differentand denote ligands with at least two nitrogen-containing heterocycles inwhich L₁, L₂ and L₃ are bound to the metal cation via nitrogen atoms, Xis a reactive functional group which is covalently bound to at least oneof the ligands L₁, L₂ and L₃ via a linker Y, n is an integer from 1 to10, and in some illustrative embodiments is 1 to 4, m is 1 or 2 and insome illustrative embodiments is 1 and A denotes the counter ion whichmay be required to equalize the charge.

The metal complex may be a luminescent metal complex i.e. a metalcomplex which undergoes a detectable luminescence reaction afterappropriate excitation. The luminescence reaction can for example bedetected by fluorescence or by electrochemiluminescence measurement. Themetal cation in this complex is for example a transition metal or a rareearth metal. The metal may be ruthenium, osmium, rhenium, iridium,rhodium, platinum, indium, palladium, molybdenum, technetium, copper,chromium or tungsten. In some illustrative embodiments ruthenium isused.

The ligands L₁, L₂ and L₃ are ligands with at least twonitrogen-containing heterocycles. Aromatic heterocycles such asbipyridyl, bipyrazyl, terpyridyl and phenanthrolyl may be used. Theligands L₁, L₂ and L₃ may be selected from bipyridine and phenanthrolinering systems.

The complex can additionally contain one or several counter ions A toequalize the charge. Examples of suitable negatively charged counterions are halogenides, OH⁻, carbonate, alkylcarboxylate, e.g.trifluoroacetate, sulphate, hexafluorophosphate and tetrafluoroborategroups. Hexafluorophosphate, trifluoroacetate and tetrafluoroborategroups are used in some illustrative embodiments. Examples of suitablepositively charged counter ions are monovalent cations such as alkalinemetal and ammonium ions.

In further embodiments the functional moiety X is an effector group. Anexemplary effector group is a therapeutically active substance.

Therapeutically active substances have different ways in which they areeffective, e.g. in inhibiting cancer. They can damage the DNA templateby alkylation, by cross-linking, or by double-strand cleavage of DNA.Other therapeutically active substances can block RNA synthesis byintercalation. Some agents are spindle poisons, such as vinca alkaloids,or anti-metabolites that inhibit enzyme activity, or hormonal andanti-hormonal agents. The effector group X may be selected fromalkylating agents, antimetabolites, antitumor antibiotics, vincaalkaloids, epipodophyllotoxins, nitrosoureas, hormonal and antihormonalagents, and toxins.

Currently exemplary alkylating agents include cyclophosphamide,chlorambucil, busulfan, Melphalan, Thiotepa, ifosphamide, Nitrogenmustard.

Currently exemplary antimetabolites include methotrexate,5-Fluorouracil, cytosine arabinoside, 6-thioguanine, 6-mercaptopurin.

Currently exemplary antitumor antibiotics include doxorubicin,daunorubicin, idorubicin, nimitoxantron, dactinomycin, bleomycin,mitomycin, and plicamycin.

Currently exemplary spindle poisons include maytansine andmaytansinoids, vinca alkaloids and epipodophyllotoxins includevincristin, vinblastin, vindestin, Etoposide, Teniposide.

Currently exemplary nitrosoureas include carmustin, lomustin, semustin,streptozocin.

Currently exemplary hormonal and antihormonal agents includeadrenocorticorticoids, estrogens, antiestrogens, progestins, aromataseinhibitors, androgens, antiandrogens.

Additional exemplary random synthetic agents include dacarbazin,hexamethylmelamine, hydroxyurea, mitotane, procarbazide, cisplastin,carboplatin.

The functional moiety X is bound either covalently or via an additionalbinding pair to at least one of (a′), (a), (b), (b′) or S. Thefunctional moiety X can occur once or several (n) times. (n) is aninteger and 1 or more than one. In some embodiments (n) is between 1 and100. Also, (n) may be 1-50. In certain embodiments n is 1 to 10, or 1 to5. In further embodiments n is 1 or 2.

For covalent binding of the functional moiety X to at least one of a′,a, b, b′ or S any appropriate coupling chemistry can be used. Theskilled artisan can easily select such coupling chemistry from standardprotocols. It is also possible to incorporate a functional moiety by useof appropriate building blocks when synthesizing a′, a, b, b′ or S.

In an exemplary embodiment functional moiety X is bound to a, b, or S ofthe binding agent as defined by Formula I. In an exemplary embodimentfunctional moiety X is bound to the spacer S of the binding agent asdefined by Formula I.

In some embodiments functional moiety X is covalently bound to a, b, orS of the binding agent as defined by Formula I.

If a functional moiety X is located within the a hybridizingoligonucleotide representing a, a′, b or b′, respectively, in some casessuch functional moiety is bound to a modified nucleotide or is attachedto the internucleosidic P atom (WO 2007/059816). Modified nucleotideswhich do not interfere with the hybridization of oligonucleotides areincorporated into those oligonucleotides. Such modified nucleotides maybe C5 substituted pyrimidines or C7 substituted 7deaza purines.

Oligonucleotides can be modified internally or at the 5′ or 3′ terminuswith non-nucleotidic entities which are used for the introduction offunctional moiety. In some embodiments such non-nucleotidic entities arelocated within the spacer S, i.e. between the two binding pair members aand b.

Many different non-nucleotidic modifier building blocks for constructionof a spacer are known in literature and a great variety is commerciallyavailable. For the introduction of a functional moiety eithernon-nucleosidic bifunctional modifier building blocks or non-nucleosidictrifunctional modified building blocks are either used as CPG forterminal labeling or as phosphroamidite for internal labeling (see:Wojczewski, C. et al., Synlett 10 (1999) 1667-1678).

Bifunctional Modifier Building Blocks

Bifunctional modifier building blocks connect a functional moiety ora—if necessary—a protected functional moiety to a phosphoramidite groupfor attaching the building block at the 5′ end (regular synthesis) or atthe 3′ end (inverted synthesis) to the terminal hydroxyl group of agrowing oligonucleotide chain.

Bifunctional modifier building blocks are, for example, non-nucleosidiccompounds. For example, such modified building blocks are C2-C18 alkyl,alkenyl, alkynyl carbon chains, whereas said alkyl, alkenyl, alkynylchains may be interrupted by additional ethyleneoxy and/or amidemoieties in order to increase hydrophilicity of the spacer and therebyof the whole linker structure. Cyclic moieties like C5-C6-cycloalkyl,C4N, C5N, C4O, C5O-heterocycloalkyl, phenyl which are optionallysubstituted with one or two C1-C6 alkyl groups can also be used asnon-nucleosidic bifunctional modified building blocks. In some casesmodified bifunctional building blocks comprise C3-C6 alkyl moieties andtri- to hexa-ethyleneglycol chains. Non-limiting examples ofbifunctional modifier building blocks are given in Table II below.

TABLE II Bifunctional non-nucleosidic modifier building blockIntroduction of Reference

 

Pon, R.T., Tetrahedron Letters 32 (1991) 1715- 1718 Theisen, P. et al.,Nucleic Acids Symposium Series (1992), 27 (Nineteenth Symposium onNucleic Acids Chemistry) 99-100 EP 0 292 128

EP 0 523 978

Meyer, A. et al., Journal of Organic Chemistry 75 (2010) 3927-3930

Morocho, A.M. et al., Nucleosides, Nucleotides & Nucleic Acids 22 (2003)1439-1441

Cocuzza, A.J., Tetrahedron Letters 30 (1989) 6287- 6290

Trifunctional Modifier Building Blocks

Trifunctional building blocks connect (i) a functional moiety or a—ifnecessary—a protected functional moiety, (ii) a phosphoramidite groupfor coupling the reporter or the functional moiety or a—if necessary—aprotected functional moiety, during the oligonucleotide synthesis to ahydroxyl group of the growing oligonucleotide chain and (iii) a hydroxylgroup which is protected with an acid labile protecting group, forexample, with a dimethoxytrityl protecting group. After removal of thisacid labile protecting group a hydroxyl group is liberated which canreact with further phosphoramidites. Therefore trifunctional buildingblocks allow for positioning of a functional moiety to any locationwithin an oligonucleotide. Trifunctional building blocks are also aprerequisite for synthesis using solid supports, e.g. controlled poreglass (CPG), which are used for 3′ terminal labeling ofoligonucleotides. In this case, the trifunctional building block isconnected to a functional moiety or a—if necessary—a protectedfunctional moiety via an C2-C18 alkyl, alkenyl, alkinyl carbon chains,whereas said alkyl, alkenyl, alkylnyl chains may be interrupted byadditional ethyleneoxy and/or amide moieties in order to increasehydrophilicity of the spacer and thereby of the whole linker structureand comprises a hydroxyl group which is attached via a cleavable spacerto a solid phase and a hydroxyl group which is protected with an acidlabile protecting group. After removal of this protecting group ahydroxyl group is liberated which could then react with aphosphoramidite.

Trifunctional Building Blocks May be Non-Nucleosidic or Nucleosidic.

Non-nucleosidic trifunctional building blocks are C2-C18 alkyl, alkenyl,alkynyl carbon chains, whereas said alkyl, alkenyl, alkynyl areoptionally interrupted by additional ethyleneoxy and/or amide moietiesin order to increase hydrophilicity of the spacer and thereby of thewhole linker structure. Other trifunctional building blocks are cyclicgroups like C5-C6-cycloalkyl, C4N, C5N, C4O, C5O heterocycloalkyl,phenyl which are optionally substituted with one ore two C1-C6 alkylgroups. Cyclic and acyclic groups may be substituted with one—(C1-C18)alkyl-O-PG group, whereas said C1-C18 alkyl comprises(Ethyleneoxy)n, (Amide)m moieties with n and m independently from eachother=0-6 and PG is an acid labile protecting group. Exemplarytrifunctional building blocks are C3-C6 alkyl, cycloalkyl, C5Oheterocycloalkyl moieties optionally comprising one amide bond andsubstituted with a C1-C6 alkyl O-PG group, wherein PG is an acid labileprotecting group, such as monomethoxytrityl, dimethoxytrityl, pixyl, andxanthyl.

Non-limiting examples for non-nucleosidic trifunctional building blocksare e.g. summarized in Table III.

TABLE III Examples for non-nucleosidic trifunctional modifier buildingBlocks Trifunctional Introduction of Reference

 

Nelson, P.S. et al., Nucleic Acids Research 20 (1992) 6253- 6259 EP 0313 219 U.S. Pat. No. 5,585,481 U.S. Pat. No. 5,451,463 EP 0 786 468 WO92/11388 WO 89/02439

 

Su, S.-H. et al., Bioorganic & Medicinal Chemistry Letters 7 (1997)1639-1644 WO 97/43451

 

Putnam, W.C. et al., Nucleosides, Nucleotides & Nucleic Acids 24 (2005)1309- 1323 US 2005/214833 EP 1 186 613

 

EP 1 431 298

WO 94/04550 Vu, H., et al., Nucleic Acids Symposium Series (1993), 29(Second International Symposium on Nucleic Acids Chemistry), 19- 20

WO 2003/019145

Behrens, C. and Dahl, O., Nucleosides & Nucleotides 18 (1999) 291-305 WO97/05156

Prokhorenko, I.A. et al., Bioorganic & Medicinal Chemistry Letters 5(1995) 2081-2084 WO 2003/104249

U.S. Pat. No. 5,849,879

Nucleosidic Modifier Building Blocks:

Nucleosidic modifier building blocks are used for internal labelingwhenever it is necessary not to influence the oligonucleotidehybridization properties compared to a non-modified oligonucleotide.Therefore nucleosidic building blocks comprise a base or a base analogwhich is still capable of hybridizing with a complementary base. Thegeneral formula of a labeling compound for labeling a nucleic acidsequence of one or more of a, a′, b, b′ or S comprised in a bindingagent according to Formula I of the present disclosure is given inFormula II.

wherein PG is an acid labile protecting group such as monomethoxytrityl,dimethoxytrityl, pixyl, and xanthyl, wherein Y is C2-C18 alkyl, alkenylalkinyl, wherein said alkyl, alkenyl, alkinyl may comprise ethyleneoxyand/or amide moieties, wherein Y is C4-C18 alkyl, alkenyl or alkinyl andcontains one amide moiety and wherein X is a functional moiety.

Specific positions of the base may be chosen for such substitution tominimize the influence on hybridization properties. Therefore thefollowing positions may be used for substitution: a) with natural bases:Uracil substituted at C5; Cytosine substituted at C5 or at N4; Adeninesubstituted at C8 or at N6 and Guanine substituted at C8 or at N2 and b)with base analogs: 7 deaza A and 7 deaza G substituted at C7; 7 deaza 8Aza A and 7 deaza 8 Aza G substituted at C7; 7 deaza Aza 2 amino Asubstituted at C7; Pseudouridine substituted at N1 and Formycinsubstituted at N2.

Non-limiting examples for nucleosidic trifunctional building blocks aregiven in Table IV.

TABLE IV Trifunctional nucleosidic A Reference

Roget, A. et al., Nucleic Acids Research 17 (1989) 7643- 7651 WO89/12642 WO 90708156 WO 93705060

Silva, J.A. et al., Biotecnologia Aplicada 15 (1998) 154-158

U.S. Pat. No. 6,531,581 EP 0 423 839

U.S. Pat. No. 4,948,882 U.S. Pat. No. 5,541,313 U.S. Pat. No. 5,817,786

WO 2001/042505

McKeen, C.M. et al., Organic & Biomolecular Chemistry 1 (2003), 2267-2275

Ramzaeva, N. et al., Helvetica Chimica Acta 83 (2000) 1108- 1126

In Tables II, III and IV, one of the terminal oxygen atom of abifunctional moiety or one of the terminal oxygen atoms of atrifunctional moiety is part of a phosphoramidite that is not shown infull detail but obvious to the skilled artisan. The second terminaloxygen atom of trifunctional building block is protected with an acidlabile protecting group PG, as defined for Formula II above.

Post-synthetic modification is another strategy for introducing acovalently bound functional moiety into a linker or a spacer molecule.In this approach an amino group is introduced by using bifunctional ortrifunctional building block during solid phase synthesis. Aftercleavage from the support and purification of the amino modifiedoligonucleotide is reacted with an activated ester of a functionalmoiety or with a bifunctional reagent wherein one functional group is anactive ester. Exemplary active esters are NHS ester or pentafluor phenylesters.

Post-synthetic modification is especially useful for introducing afunctional moiety which is not stable during solid phase synthesis anddeprotection. Examples are modification withtriphenylphosphincarboxymethyl ester for Staudinger ligation (Wang, C.C. et al., Bioconjugate Chemistry 14 (2003) 697-701), modification withdigoxigenin or for introducing a maleinimido group using commercialavailable sulfo SMCC.

The functional moiety X in one embodiment is bound to at least one ofa′, a, b, b′ or S via an additional binding pair.

The additional binding pair to which a functional moiety X can be boundmay be a leucine zipper domain or a hybridizing nucleic acid. In casethe functional moiety X is bound to at least one of a′, a, b, b′ or Svia an additional binding pair member, the binding pair member to whichX is bound and the binding pairs a′:a and b:b′, respectively, all areselected to have different specificity. The binding pairs a:a′, b:b′ andthe binding pair to which X is bound each bind to (e.g. hybridize with)their respective partner without interfering with the binding of any ofthe other binding pairs.

Covalent Coupling of a Member of a Binding Pair to a Monovalent Binder

Depending on the biochemical nature of the binder different conjugationstrategies are at hand.

In case the binder is a naturally occurring protein or a recombinantpolypeptide of between 50 to 500 amino acids, there are standardprocedures in text books describing the chemistry for synthesis ofprotein conjugates, which can be easily followed by the skilled artisan.(Hackenberger, C. P. et al., Angew. Chem., Int. Ed., 47(2008)10030-10074).

In one embodiment the reaction of a maleinimido moiety with a cysteinresidue within the protein is used. This is an exemplary couplingchemistry in case e.g. an Fab or Fab′-fragment of an antibody is used amonovalent binder. Alternatively in one embodiment coupling of a memberof a binding pair (a′ or b′, respectively, of Formula I) to theC-terminal end of the binder polypeptide is performed. C-terminualmodification of a protein, e.g. of an Fab-fragment can e.g. be performedas described (Sunbul, Murat and Yin, Jun, Organic & BiomolecularChemistry 7 (2009) 3361-3371).

In general site specific reaction and covalent coupling of a bindingpair member to a monovalent polypeptidic binder is based on transforminga natural amino acid into an amino acid with a reactivity which isorthogonal to the reactivity of the other functional groups present in aprotein. For example, a specific cystein within a rare sequence contextcan be enzymatically converted in an aldehyde (see Frese, M.-A. et al.,ChemBioChem 10 (2009) 425-427). It is also possible to obtain a desiredamino acid modification by utilizing the specific enzymatic reactivityof certain enzymes with a natural amino acid in a given sequence context(see e.g.: Taki, M. et al., Protein Engineering, Design & Selection 17(2004) 119-126; Gautier, A. et al., Chemistry & Biology 15 (2008)128-136; Protease-catalyzed formation of C—N bonds is used by Bordusa,F., Highlights in Bioorganic Chemistry (2004) 389-403 andSortase-mediated protein ligation is used by Mao, H. et al., in J. Am.Chem. Soc. 126 (2004) 2670-2671 and reviewed by Proft, T., inBiotechnol. Lett 32 (2010) 1-10).

Site specific reaction and covalent coupling of a binding pair member toa monovalent polypeptidic binder can also be achieved by the selectivereaction of terminal amino acids with appropriate modifying reagents.

The reactivity of an N-terminal cystein with benzonitrils (Ren, Hongjun,Xiao, et al., Angewandte Chemie, International Edition 48 (2009)9658-9662) can be used to achieve a site-specific covalent coupling.

Native chemical ligation can also rely on C-terminal cystein residues(Taylor, E. Vogel, Imperiali, B., Nucleic Acids and Molecular Biology 22(2009) (Protein Engineering) 65-96).

EP 1 074 563 describes a conjugation method which is based on the fasterreaction of a cystein within a stretch of negatively charged amino acidswith a cystein located in a stretch of positively charged amino acids.

The monovalent binder may also be a synthetic peptide or peptide mimic.In case a polypeptide is chemically synthesized, amino acids withorthogonal chemical reactivity can be incorporated during such synthesis(de Graaf, A. J. et al., Bioconjugate Chemistry 20 (2009) 1281-1295).Since a great variety of orthogonal functional groups is at stake andcan be introduced into a synthetic peptide, conjugation of such peptideto a linker is standard chemistry.

In order to obtain a mono-labeled protein the conjugate with 1:1stoichiometry may be separated by chromatography from other conjugationproducts. This procedure is facilitated by using a dye labeled bindingpair member and a charged spacer. By using this kind of labeled andhighly negatively charged binding pair member, mono conjugated proteinsare easily separated from non labeled protein and proteins which carrymore than one linker, since the difference in charge and molecularweight can be used for separation. The fluorescent dye is valuable forpurifying the binding agent from un-bound components, like a labeledmonovalent binder.

Therefore in one embodiment a binding pair member (a′ and/or b′,respectively of Formula I) which is labeled with a fluorescent dye (e.g.synthesized using a bifunctional or trifunctional modifier buildingblock in combination with bifunctional spacer building blocks duringsynthesis) for forming the binding agent of the present disclosure maybe used. In an exemplary embodiment the spacer S as well as thesequences a, a′, b and b′ are DNA and at least one of a′ or b′,respectively, is labeled with a fluorescent dye. In some embodiments thespacer S as well as the sequences a, a′, b and b′ are DNA and both a′and b′, respectively, are labeled each with a different fluorescent dye.

In one embodiment the present disclosure relates to a bispecific bindingagent of the Formula I: A-a′:a-S-b:b′-B:X(n); wherein A as well as B isa monovalent specific binder, wherein a′:a as well as b:b′ represent abinding pair with a′:a and b:b′ having a different specificity, whereinS represents a spacer, wherein (: X) denotes a functional moiety boundvia a further binding pair to at least one of a′, a, b, b′ or S, wherein(n) is an integer and at least 1, wherein - represents a covalent bond,and wherein the linker a-S-b has a length of 6 to 100 nm.

In some embodiments the binding pairs a′:a and b:b′ are hybridizingnucleic acid sequences, the spacer S is a nucleic acid and the furtherbinding pair to which the functional moiety X is bound is also a nucleicacid. In such embodiments the spacer S may be construed to comprise, inaddition to the two specifically hybridizing sequences a and a′ and band b′, respectively, one or more further sequences also capable ofhybridizing to its or their complementary sequences. In this embodimenta functional moiety X is bound to the spacer S via a further bindingpair also consisting of hybridizing nucleic acid sequences.

A monovalent binder for use in the construction of a binding agent asdisclosed herein has to have a Kdiss from 10⁻²/sec to 10⁻⁵/sec. Also, amonovalent binder for use in the construction of a binding agent asdisclosed herein has to have a Kdiss from 10⁻³/sec to 10⁻⁵/sec.

According to some embodiments, in the binding agent according to FormulaI, each of the monovalent binders A and B, respectively has a Kdiss from10⁻²/sec to 10⁻⁵/sec and in some illustrative embodiments from 10⁻³/secto 10⁻⁵/sec.

In some embodiments, the binding agent according to Formula I has aKdiss of 10⁻⁵/sec or better, or may have a Kdiss of 10⁻⁶/sec or better.In some embodiments the binding agent according to Formula I may have aKdiss of 10⁻⁷/sec or better.

As the skilled artisan will appreciate the Kdiss is atemperature-dependent value. Logically, the Kdiss-values of a bindingagent according to the present disclosure are determined at the sametemperature. As will be appreciated, a Kdiss-value is determined at thesame temperature at which the binding agent shall be used, e.g., anassay shall be performed. In one embodiment the Kdiss-values areestablished at room temperature, i.e. at 20° C., 21° C., 22° C., 23° C.,24° C. or 25° C., respectively. In one embodiment the Kdiss-values areestablished at 4 or 8° C., respectively. In one embodiment theKdiss-values are established at 25° C. In one embodiment theKdiss-values are established at 37° C.

As mentioned already above, it is now possible and prettystraightforward to produce a binding agent as defined in Formula I. Afull library of a binding agent according to Formula I can be easilyprovided, analyzed and the most powerful binding agent out of suchlibrary produced at large scale, as required.

The library mentioned above refers to a full set of binding agentsaccording to Formula I, wherein each A, a, a′, b, b′ and B are identicaland wherein in the length of the spacer S is adjusted to best meet therequirements set out for the binding agent. It is easily possible tofirst use a spacer ladder spanning the whole spectrum of 1 to 100 nm andhaving steps that are about 10 nm apart. The spacer length is then againeasily further refined around the most appropriate length identified inthe first round.

In one embodiment the present disclosure relates to a method ofproducing a binding agent of the Formula I: A-a′:a-S-b:b′-B:X(n),wherein A as well as B is a monovalent binder, wherein a′:a as well asb:b′ is a binding pair, wherein a′ and a and do not interfere with thebinding of b to b′ and vice versa, wherein S is a spacer of at least 1nm in length, wherein (: X) denotes a functional moiety bound eithercovalently or via a binding pair to at least one of a′, a, b, b′ or S,wherein (n) is an integer and at least 1, wherein - represents acovalent bond, and wherein the linker a-S-b has a length of 6 to 100 nm,the method comprising the steps of: a) synthesizing A-a′ and b′-B,respectively, b) synthesizing the linker a-S-b and c) forming thebinding agent of Formula I, wherein the functional moiety X bound to atleast one of a′, a, b, b′ or S is bound in step a), b) or c).

In some embodiments of this method several linker molecules with spacersof various lengths are synthesized and used in the formation of bindingagents according to Formula I comprising spacers of variable length andthose binding agent(s) are selected having an improvement in the Kdissof at least 5-fold over the better of the two monovalent binders.Selection of a binding agent with the desired Kdiss in one embodiment isperformed by BiaCore-analysis as disclosed in Example 2.8.

The binding agent according to the present disclosure can e.g. be usedto strongly bind an analyte in an immunoassay. If e.g. an analyte has atleast two non-overlapping epitopes the binding agent of the presentdisclosure is construed such that the spacer S has the optimal lengthfor synergistic binding of the monovalent binders to these epitopes.This improvement can e.g. be of great utility in a method for detectionof an analyte employing such binding agent. In one embodiment thepresent disclosure therefore relates to the use of a binding agent asdisclosed herein above in the detection of an analyte of interest. Incertain embodiments the detection method used is an enzyme-linkedimmunosorbent assay (ELISA), a direct, indirect, competitive or sandwichimmuno assay employing any appropriate way of signal detection, e.g.electrochemiluminescense, or the binding agent is used inimmunohistochemistry.

The following examples, sequence listing, and figures are provided forthe purpose of demonstrating various embodiments of the instantdisclosure and aiding in an understanding of the present disclosure, thetrue scope of which is set forth in the appended claims. These examplesare not intended to, and should not be understood as, limiting the scopeor spirit of the instant disclosure in any way. It should also beunderstood that modifications can be made in the procedures set forthwithout departing from the spirit of the disclosure.

Illustrative Embodiments

The following comprises a list of illustrative embodiments according tothe instant disclosure which represent various embodiments of theinstant disclosure. These illustrative embodiments are not intended tobe exhaustive or limit the disclosure to the precise forms disclosed,but rather, these illustrative embodiments are provided to aide infurther describing the instant disclosure so that others skilled in theart may utilize their teachings.

1. A binding agent of the Formula A-a′:a-S-b:b′-B:X(n), wherein A aswell as B is a monovalent binder, wherein a′:a as well as b:b′ is abinding pair wherein a′ and a do not interfere with the binding of b tob′ and vice versa, wherein S is a spacer of at least 1 nm in length,wherein (: X) denotes a functional moiety bound either covalently or viaa binding pair to at least one of a′, a, b, b′ or S, wherein (n) is aninteger and at least 1, wherein - represents a covalent bond, andwherein the linker a-S-b has a length of 6 to 100 nm.2. The binding agent of embodiment 1, wherein the spacer S is 1 to 95 nmin length.3. The binding agent of embodiments 1 or 2, wherein the binding pairsare selected from the group consisting of leucine zipper domain dimersand hybridizing nucleic acid sequences.4. The binding agent of any of embodiments 1 to 3, wherein the bindingpairs both are hybridizing nucleic acid sequences and wherein thedifferent hybridizing nucleic acid sequences of the binding pairs a′:aand b:b′ do not hybridize with one another.5. The binding agent of any of embodiments 1 to 4, wherein the spacer Sis a nucleic acid.6. The binding agent of any of embodiments 1 to 5, wherein the spacer Sis a nucleic acid and wherein both binding pairs a′:a as well as b:b′also are a nucleic acid.7. The binding agent of any of embodiments 1 to 6, wherein the spacer Sis a nucleic acid and wherein both binding pairs a′:a as well as b:b′are nucleic acids and wherein the monovalent binders A and B are nucleicacids.8. The binding agent of any of embodiments 1 to 7, wherein X is afunctional moiety selected from the group consisting of a labelinggroup, a binding group and an effector group9. The binding agent of any of embodiments 1 to 8, wherein thefunctional moiety X is bound to a, b, or S.10. The binding agent of any of embodiments 1 to 9, wherein thefunctional moiety X is bound to the spacer S.11. The binding agent of any embodiments 1 to 10, wherein the functionalmoiety X is covalently bound to the spacer S.12. The binding agent of any of embodiments 1 to 10, wherein thefunctional moiety X is bound to the spacer S via a hybridizing nucleicacid.13. The binding agent of any of embodiments 1 to 3, or embodiments 8 to12, wherein the monovalent binders A and B are polypeptides such asFab-fragments of monoclonal antibodies.14. Use of a binding agent according to any of embodiments 1 to 13 inthe detection of an analyte of interest.15. Use of a binding agent according to any of embodiments 1 to 13 in animmuno assay.

EXAMPLES Bi-Valent Binding Agent to Troponin T

1.1 Monoclonal Antibodies and Fab′-Fragments

Two monoclonal antibodies binding to human cardiac Troponin T atdifferent, non-overlapping epitopes, epitope A′ and epitope B′,respectively, were used. Both these antibodies are used in the currentRoche Elecsys™ Troponin T assay, wherein Troponin T is detected in asandwich immuno assay format.

Purification of the monoclonal antibodies from culture supernatant wascarried out using state of the art methods of protein chemistry.

The purified monoclonal antibodies are protease digested with eitherpre-activated papain (anti-epitope A′ MAb) or pepsin (anti-epitope B′MAb) yielding F(ab′)2 fragments that are subsequently reduced toFab′-fragments, i.e. A and B, respectively, in Formula I(A-a′:a-S-b:b′-B:X_(n)), with a low concentration of cysteamin at 37°C.,. The reaction is stopped by separating the cysteamin on a SephadexG-25 column (GE Healthcare) from the polypeptide-containing part of thesample.

1.2 Conjugation of Fab′-Fragments to ssDNA-Oligonucleotides

The Fab′-fragments are conjugated with the below described activatedssDNAa and ssDNAb oligonucleotides.

Preparation of the Fab′-fragment-ssDNA conjugates A″ and B″,respectively:

a) Fab′-Anti-Troponin T<Epitope a′>-ssDNA-Conjugate (=a″)

For preparation of the Fab′-anti-Troponin T<epitope A′>-ssDNAa-conjugateA″ a derivative of SED ID NO:5 is used, i.e. 5′-AGT CTA TTA ATG CTT CTGC(=SEQ ID NO:5)-XXX-Y-Z-3′, wherein X=propylene-phosphate introduced viaPhosphoramidite C3(3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research), wherein Y=3″-Amino-Modifier C6 introduced via 3′-AminoModifier TFA Amino C-6 Icaa CPG (ChemGenes) and whereinZ=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced viaSulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(ThermoFischer).

b) Fab′-Anti-Troponin T<Epitope B′>-ssDNAb-Conjugate (=B″)

For the preparation of the Fab′-anti-Troponin T<epitopeB′>-ssDNA-conjugate (B″) a derivative of SEQ ID NO:6 is used, i.e.5′-Y-Z-XXX-AGT TCT ATC GTC GTC CA-3′, wherein X=propylene-phosphateintroduced via Phosphoramidite C3(3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research), wherein Y=5′-Amino-Modifier C6 introduced via(6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(Glen Research), and whereinZ=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced viaSulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(ThermoFischer).

The oligonucleotides of SEQ ID NO:5 or 6, respectively, have beensynthesized by state of the art oligonucleotide synthesis methods. Theintroduction of the maleinimido group was done via reaction of the aminogroup of Y with the succinimidyl group of Z which was incorporatedduring the solid phase oligonucleotide synthesis process.

The single-stranded DNA constructs shown above bear a thiol-reactivemaleimido group that reacts with a cysteine of the Fab′ hinge regiongenerated by the cysteamine treatment. In order to obtain a highpercentage of single-labeled Fab′-fragments the relative molar ratio ofssDNA to Fab′-fragment is kept low. Purification of single-labeledFab′-fragments (ssDNA:Fab′=1:1) occurs via anion exchange chromatography(column: MonoQ, GE Healthcare). Verification of efficient labeling andpurification is achieved by analytical gel filtration chromatography andSDS-PAGE.

1.3 Biotinylated Linker Molecules

The oligonucleotides used in the ssDNA linkers L1, L2 and L3,respectively, have been synthesized by state of the art oligonucleotidesynthesis methods and employing a biotinylated phosphoramidite reagentfor biotinylation.

Linker 1 (=L1), a biotinylated ssDNA linker 1 with no spacer exceptbiotinylated thymidine has the following composition: 5-GCA GAA GCA TTAATA GAC T (Biotin-dT)-TGG ACG ACG ATA GAA CT-3′. It comprises ssDNAoligonucleotides of SEQ ID NO:7 and 8, respectively, and wasbiotinylated by using Biotin-dT (=T-Bi)(5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research) in the middle of the spacer.

Linker 2 (=L2), a biotinylated ssDNA linker 2 with a 11 mer spacer hasthe following composition: 5-GCA GAA GCA TTA ATA GAC T T5-(Biotin-dT)-T5TGG ACG ACG ATA GAA CT-3′. It comprises ssDNA oligonucleotides of SEQ IDNO:7 and 8, respectively, twice oligonucleotide stretches of fivethymidines each and was biotinylated by using Biotin-dT(5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research) in the middle of the spacer.

Linker 3 (=L3), a biotinylated ssDNA linker 3 with a 31 mer spacer hasthe following composition: 5-GCA GAA GCA TTA ATA GAC TT15-(Biotin-dT)-T15 TGG ACG ACG ATA GAA CT-3′. It comprises ssDNAoligonucleotides of SEQ ID NO:7 and 8, respectively, twiceoligonucleotide stretches of fifteen thymidines each and wasbiotinylated by using Biotin-dT(5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research).

1.4 Epitopes for Monovalent Troponin T Binders A and B, Respectively

Synthetic peptides have been construed that individually only have amoderate affinity to the corresponding Fab′-fragment derived from theanti-Troponin T antibodies a and b, respectively.

a) The Epitope a′ for Antibody a is Comprised in:

SEQ ID NO:9=ERAEQQRIRAEREKEUUSLKDRIEKRRRAERAEamide, wherein U representsβ-Alanin.

b) The Epitope B′ for Antibody b is Comprised in:

SEQ ID NO:10=SLKDRIERRRAERAEOOERAEQQRIRAEREKEamide, wherein O representsAmino-trioxa-octanoic-acid

As the skilled artisan will appreciate it is possible to combine thesetwo epitope-containing peptides in two ways and both variants have beendesigned and prepared by linear combining the epitopes A′ and B′. Thesequences of both variants, the linear sequences of epitopes A′-B′(=TnT-1) and B′-A′ (=TnT-2), respectively have been prepared by state ofthe art peptide synthesis methods.

The sequences for epitopes A′ and B′, respectively, had been modifiedcompared to the original epitopes on the human cardiac Troponin Tsequence (P45379/UniProtKB) in order to reduce the binding affinity foreach of the Fabs thereto. Under these circumstances the dynamics of theeffect of hetero-bi-valent binding is better visible, e.g. by analyzingbinding affinity with the Biacore™ Technology.

1.5 Biomolecular Interaction Analysis

For this experiment a Biacore™ 3000 instrument (GE Healthcare) was usedwith a Biacore™ SA sensor mounted into the system at T=25° C.Preconditioning was done at 100 μl/min with 3×1 min injection of 1 MNaCl in 50 mM NaOH and 1 min 10 mM HCl.

HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20 wasused as system buffer. The sample buffer was identical to the systembuffer.

The Biacore™ 3000 System was driven under the control software V1.1.1.Flow cell 1 was saturated with 7 RU D-biotin. On flow cell 2, 1063 RUbiotinylated ssDNA linker L1 was immobilized. On flow cell 3, 879 RUbiotinylated ssDNA linker L2 was immobilized. On flow cell 4, 674 RUbiotinylated ssDNA linker L3 was captured.

Thereafter, Fab′ fragment DNA conjugate A″ was injected at 600 nM. Fab′fragment DNA conjugate B″ was injected into the system at 900 nM. Theconjugates were injected for 3 min at a flow rate of 2 μl/min. Theconjugates were consecutively injected to monitor the respectivesaturation signal of each Fab′ fragment DNA conjugate on its respectivelinker. Fab′ combinations were driven with a single Fab′ fragment DNAconjugate A″, a single Fab′ fragment DNA conjugate B″ and both Fab′fragment DNA conjugates A″ and B″ present on the respective linker.Stable baselines were generated after the linkers have been saturated bythe Fab′ fragment DNA conjugates, which was a prerequisite for furtherkinetic measurements.

The artificial peptidic analytes TnT-1 and TnT-2 were injected asanalytes in solution into the system in order to interact with thesurface presented Fab′ fragments.

TnT-1 was injected at 500 nM, TnT-2 was injected at 900 nM analyteconcentration. Both peptides were injected at 50 μl/min for 4 minassociation time. The dissociation was monitored for 5 min. Regenerationwas done by a 1 min injection at 50 μl/min of 50 mM NaOH over all flowcells.

Kinetic data was determined using the Biaevaluation software (V.4.1).The dissociation rate kd (1/s) of the TnT-1 and TnT-2 peptides from therespective surface presented Fab′ fragment combinations was determinedaccording to a linear Langmuir 1:1 fitting model. The complex halftimein min were calculated according to the solution of the first orderkinetic equation: In(2)/(60*kd).

Results:

The experimental data given in Tables 1 and 2, respectively demonstratean increase in complex stability between analyte (TnT-1 or TnT-2),respectively, and the various heterobi-valent Fab′-Fab′ dimers A″-B″ ascompared to the monovalent dsDNA Fab′ A″ or B″ conjugate, respectively.This effect is seen in each Table in line 1 compared to lines 2 and 3.

TABLE 1 Analysis data using TnT-1 with linkers of various length Fab′fragment Fab′ fragment kd t½ diss DNA conjugate A″ DNA conjugate B″(1/s) (min) a) Linker L1 x x 6.6E−03 1.7 x — 3.2E−02 0.4 — x 1.2E−01 0.1b) Linker L2 x x  4.85E−03 2.4 x — 2.8E−02 0.4 — x 1.3E−01 0.1 c) linkerL3 Fab′ fragment Fab′ fragment kd t½ diss DNA conjugate A″ DNA conjugateB″ (/1/s) (min) x x 2.0E−03  5.7 x — 1.57E−02 0.7 — x 1.56E−02 0.7

TABLE 2 Analysis data using TnT-2 with linkers of various length Fab′fragment Fab′ fragment kd t½ diss DNA conjugate A″ DNA conjugate B″(/1/s) (min) a) Linker L1 x x 1.4E−02 0.8 x — 4.3E−02 0.3 — x 1.4E−010.1 b) Linker L2 x x 4.9E−03 2.3 x — 3.5E−02 0.3 — x 1.3E−01 0.1 c)Linker L3 x x 8.0E−03 1.5 x — 4.9E−02 0.2 — x 3.2E−01 0.04

The avidity effect is further dependent on the length of the linker. Inthe sub-tables shown under Table 1, i.e. for the artificial analyteTnT-1, the linker L3 comprising a 31 mer thymidine-based spacer showsthe lowest dissociation rate or highest complex stability.

In the sub-tables shown under Table 2 the linker L2 comprising an 11 merthymidine-based spacer exhibits the lowest dissociation rate or highestcomplex stability for the artificial analyte TnT-2.

These data taken together demonstrate that the flexibility in linkerlength as inherent to the approach given in the present disclosure is ofgreat utility and advantage.

Example 2 Bi-Valent Binding Agent to Phosphorylated IGF-1R

2.1 Monoclonal Antibody Development (mAb 8.1.2 and mAb 1.4.168)

a) Immunization of Mice

BALB/C mice are immunized at week 0, 3, 6 and 9, respectively. Perimmunization 100 μg of the conjugate comprising the phosphorylatedpeptide pIGF-1R (1340-1366) (SEQ ID NO:11) is used. This peptide hadbeen phosphorylated at tyrosine 1346 (=1346-pTyr) and coupled to KLH viathe C-terminal cysteine (=Aoc-Cys-MP-KLH-1340) to yield the conjugateused for immunization. At weeks 0 and 6, respectively, the immunizationis carried out intraperitoneally and at weeks 3 and 9, respectively,subcutaneously at various parts of the mouse body.

b) Fusion and Cloning

Spleen cells of immunized mice are fused with myeloma cells according toGalfre G., and Milstein C., Methods in Enzymology 73 (1981) 3-46. Inthis process ca 1×10⁸ spleen cells of an immunized mouse are mixed with2×10⁷ myeloma cells a(P3×63-Ag8653, ATCC CRL1580) and centrifuged (10min at 250 g and 37° C.). The cells are then washed once with RPMI 1640medium without fetal calf serum (FCS) and centrifuged again at 250 g ina 50 ml conical tube. The supernatant is discarded, the cell sediment isgently loosened by tapping, 1 ml PEG (molecular weight 4000, Merck,Darmstadt) is added and mixed by pipetting. After 1 min incubation in awater bath at 37° C., 5 ml RPMI 1640 without FCS is added drop-wise atroom temperature within a period of 4-5 min. This step is repeated withadditional 10 ml RPMI 1640 without FCS. Afterwards 25 ml RPMI 1640containing 10% FCS is added followed by an incubation step at 37° C., 5%CO₂ for 30 minutes. After centrifugation for 10 min at 250 g and 4° C.the sedimented cells are taken up in RPMI 1640 medium containing 10% FCSand seeded out in hypoxanthine-azaserine selection medium (100 mmol/lhypoxanthine, 1 pg/ml azaserine in RPMI 1640+10% FCS). Interleukin 6 at100 U/ml is added to the medium as a growth factor. After 7 days themedium is exchanged with fresh medium. On day 10, the primary culturesare tested for specific antibodies. Positive primary cultures are clonedin 96-well cell culture plates by means of a fluorescence activated cellsorter.

c) Immunoglobulin Isolation from the Cell Culture Supernatants

The hybridoma cells obtained are seeded out at a density of 1×10⁷ cellsin CELLine 1000 CL flasks (Integra). Hybridoma cell supernatantscontaining IgGs are collected twice a week. Yields typically rangebetween 400 μg and 2000 μg of monoclonal antibody per 1 ml supernatant.Purification of the antibody from culture supernatant was carried outusing conventional methods of protein chemistry (e.g. according toBruck, C., Methods in Enzymology 121 (1986) 587-695).

2.2 Synthesis of Hybridizable Oligonucleotides

The following amino modified precursors, comprising the sequences givenin SEQ ID NOs: 5 and 6, respectively, were synthesized according tostandard methods. The below given oligonucleotides not only comprise theso-called aminolinker, but also a fluorescent dye. As the skilledartisan will readily appreciate, this fluorescent dye is very convenientto facilitate purification of the oligonucleotide as such, as well as ofcomponents comprising them.

a) 5′-Fluorescein-AGT CTA TTA ATG CTT CTG C-(SpacerC3)3-C7-Aminolinker-; b) 5-Cy5 AGT CTA TTA ATG CTT CTG C-(SpacerC3)3-C7-Aminolinker-; c) 5′-Aminolinker-(Spacer C3)3-AGT TCT ATC GTC GTCCA-Fluorescein-3′;

d) 5′-Fluorescein-(beta L AGT CTA TTA ATG CTT CTG C)-(SpacerC3)3-C7-Aminolinker-; (beta L indicates that this is an L-DNAoligonucleotide) ande) 5′-Aminolinker-(Spacer C3)3-(beta L-AGT TCT ATC GTC GTCCA)-Fluorescein-3′ (beta L indicates that this is an L-DNAoligonucleotide).

Synthesis was performed on an ABI 394 synthesizer at a 10 μmol scale inthe trityl on (for 5′ amino modification) or trityl off mode (for 3′amino modification) using commercially available CPGs as solid supportsand standard dA(bz), dT, dG (iBu) and dC(Bz) phosphoramidites (SigmaAldrich).

The following amidites, amino modifiers and CPG supports were used tointroduce the C3-spacer, a dye and amino moieties, respectively, duringoligonucleotide synthesis:

Spacer Phosphoramidite C3(3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research);

5′ amino modifier is introduced by using 5′-Amino-Modifier C6(6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(Glen Research);

5′-Fluorescein Phosphoramidite6-(3′,6′-dipivaloylfluoresceinyl-6-carboxamido)-hexyl-1-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(Glen Research);

Cy5™ Phosphoramidite1-[3-(4-monomethoxytrityloxy)propyl]-1′-[3-[(2-cyanoethyl)-(N,N-diisopropylphosphoramidityl]propyl]-3,3,3′,3′-tetramethylindodicarbocyaninechloride (Glen Research);

LightCycler Fluoresceine CPG 500 A (Roche Applied Science); and

3′-Amino Modifier TFA Amino C-6 Icaa CPG 500 A (Chemgenes),

For Cy5 labeled oligonucleotides, dA(tac), dT, dG(tac) dC(tac)phosphoramidites, (Sigma Aldrich), were used and deprotection with 33%ammonia was performed for 2 h at room temperature.

L-DNA oligonucleotides were synthesized by using beta-L-dA(bz), dT, dG(iBu) and dC(Bz) phosphoramidites (Chemgenes)

Purification of fluorescein modified hybridizable oligonucleotides wasperformed by a two step procedure: First the oligonucleotides werepurified on reversed-phase HPLC (Merck-Hitachi-HPLC; RP-18 column;gradient system [A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN]: 3min, 20% B in A, 12 min, 20-50% B in A and 25 min, 20% B in A with aflow rate of 1.0 ml/min, detection at 260 nm. The fractions (monitoredby analytical RP HPLC) containing the desired product were combined andevaporated to dryness. (Oligonucleotides modified at the 5′ end withmonomethoxytrityl protected alkylamino group are detriylated byincubating with 20% acetic acid for 20 min). The oligomers containingfluorescein as label were purified again by IEX chromatography on a HPLC[Mono Q column: Buffer A: Sodium hydroxide (10 mM/I; pH ˜12) Buffer B 1MSodium chloride dissolved in Sodium hydroxide (10 mM/I; pH ˜12)gradient: in 30 minutes from 100% buffer A to 100% buffer B flow 1ml/min detection at 260 nm]. The product was desalted via dialysis.

Cy5 labeled oligomers were used after the first purification onreversed-phase HPLC (Merck-Hitachi-HPLC; RP-18 column; gradient system[A: 0.1 M (Et3NH)OAc (pH 7.0)/MeCN 95:5; B: MeCN]: 3 min, 20% B in A, 12min, 20-50% B in A and 25 min, 20% B in A with a flow rate of 1.0ml/min, detection at 260 nm. The oligomers were desalted by dialysis andlyophilized on a Speed-Vac evaporator to yield solids which were frozenat −24° C.

2.3 Activation of Hybridizable Oligonucleotides

The amino modified oligonucleotides from Example 2 were dissolved in 0.1M sodium borate buffer pH 8.5 buffer (c=600 μmol) and reacted with a18-fold molar excess of Sulfo SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate dissolved in DMF (c=3mg/100 μl) from Thermo Scientific, The reaction product was thoroughlydialyzed against water in order to remove the hydrolysis product ofsulfoSMCC 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

The dialysate was concentrated by evaporation and directly used forconjugation with a monovalent binder comprising a thiol group.

2.4 Synthesis of Linker Oligonucleotides Comprising HybridizableOligonucleotides at Both Ends

Oligonucleotides were synthesized by standard methods on an ABI 394synthesizer at a 10 μmol scale in the trityl on mode using commerciallyavailable dT-CPG as solid supports and using standard dA(bz), dT, dG(iBu) and dC(Bz) phosphoramidites (Sigma Aldrich).

L-DNA oligonucleotides were synthesized by using commercially availablebeta L-dT-CPG as solid support and beta-L-dA(bz), dT, dG (iBu) anddC(Bz) phosphoramidites (Chemgenes)

Purification of the oligonucleotides was performed as described underExample 3 on a reversed-phase HPLC. The fractions (analyzed/monitored byanalytical RP HPLC) containing the desired product were combined andevaporated to dryness. Detriylation was performed by incubating with 80%acetic acid for 15 min) The acetic acid was removed by evaporation. Thereminder was dissolved in water and lyophilized

The following amidites and CPG supports were used to introduce the C18spacer, digoxigenin and biotin group during oligonucleotide synthesis:

Spacer Phosphoramidite 18 (18-O-Dimethoxytritylhexaethyleneglycol,1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (Glen Research);

Biotin-dT(5′-Dimethoxytrityloxy-5-[N-((4-t-butylbenzoyl)-biotinyl)-aminohexyl)-3-acrylimido]-2′-deoxyUridine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research);

BiotinPhosphoramidite1-Dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramiditeand

5′-Dimethoxytrityl-5-[N-(trifluoroacetylaminohexyl)-3-acrylimido]-2′-deoxyuridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite for aminomodification and postlabeling with Digoxigenin-N-Hydroxyl-succininimidylester.

The following bridging constructs or linkers were synthesized:

Linker 1: 5′-G CAG AAG CAT TAA TAG ACT-TGG ACG ACG ATA GAA CT-3′Linker 2: 5′-G CAG AAG CAT TAA TAG ACT-(T40)-TGG ACG ACG ATA GAA CT-3′Linker 3: 5′-[B-L]G CAG AAG CAT TAA TAG ACT-(Biotin-dT)-TGG ACG ACG ATAGAA CT-3′ Linker 4:5′-[B-L]G CAG AAG CAT TAA TAG ACT-T5-(Biotin-dT)-T5-TGG ACG ACGATA GAA CT-3′ Linker 5:5′-[B-L]G CAG AAG CAT TAA TAG ACT-T20-(Biotin-dT)-T20-TGG ACG ACGATA GAA CT-3′ Linker 6: 5′-[B-L]G CAG AAG CAT TAA TAG ACT-T30-(Biotin-dT)-T30-TGG ACG ACG ATA GAA CT-3′Linker 7: 5′-GCA GAA GCA TTA ATA GAC T T5-(Biotin-dT)-T5 TG GAC GAC GATAGA ACT-3′ Linker 8:5′-GCA GAA GCA TTA ATA GAC T T10-(Biotin-dT)-T10 TGG ACG ACG ATAGAA CT-3′ Linker 9:5′-GCA GAA GCA TTA ATA GAC T T15-(Biotin-dT)-T15 TGG ACG ACG ATAGAA CT-3′ Linker 10:5′-GCA GAA GCA TTA ATA GAC T T20-(Biotin-dT)-T20 TGG ACG ACGATA GAA CT-3′ Linker 11:5′-G CAG AAG CAT TAA TAG ACT-Spacer C18- (Biotin-dT)-Spacer C18-TGG ACG ACG ATA GAA CT-3′ Linker 12:5′-G CAG AAG CAT TAA TAG ACT-(Spacer C18)2-(Biotin-dT)-(SpacerC18)2-TGG ACG ACG ATA GAA CT-3′ Linker 13:5′-G CAG AAG CAT TAA TAG ACT-(Spacer C18)3-(Biotin-dT)-(SpacerC18)3-TGG ACG ACG ATA GAA CT-3′ Linker 14:5′-G CAG AAG CAT TAA TAG ACT-(Spacer C18)4-(Biotin-dT)-(SpacerC18)4-TGG ACG ACG ATA GAA CT-3′ Linker 15:5′-G CAG AAG CAT TAA TAG ACT-T20-(Dig-dT)-T20-TGG ACG ACG ATA GAA CT-3′Linker 16: 5′-G CAG AAG CAT TAA TAG ACT-(Dig-dT)-TGG ACG ACG ATA GAA CT-3′ Linker 17:5′-G CAG AAG CAT TAA TAG ACT-(Biotin-dT)-TGG ACG ACG ATA GAA CT-3′

The above bridging construct examples comprise at least a firsthybridizable oligonucleotide and a second hybridizable oligonucleotide.Linkers 3 to 17 in addition to the hybridizable nucleic acid stretchescomprise a central biotinylated or digoxigenylated thymidine,respectively, or a spacer consisting of thymidine units of the lengthgiven above.

The 5′-hybridizable oligonucleotide corresponds to SEQ ID NO:7 and the3′-hybridizable oligonucleotide corresponds to SEQ ID NO:8,respectively. The oligonucleotide of SEQ ID NO:7 will readily hybridizewith the oligonucleotide of SED ID NO:5. The oligonucleotide of SEQ IDNO:8 will readily hybridize with the oligonucleotide of SED ID NO:6.

In the above bridging construct examples [B-L] indicates that an L-DNAoligonucleotide sequence is given; spacer C 18, Biotin and Biotin dTrespectively, refer to the C18 spacer, the Biotin and the Biotin-dT asderived from the above given building blocks; and T with a numberindicates the number of thymidine residues incorporated into the linkerat the position given.

2.5 Assembly of Dual Binder Construct

A) Cleavage of IgGs and Labeling of Fab′ Fragments with ssDNA

Purified monoclonal antibodies were cleaved with the help of pepsinprotease yielding F(ab′)2 fragments that are subsequently reduced toFab′ fragments by treatment with low concentrations of cysteamine at 37°C. The reaction is stopped via separation of cysteamine on a PD 10column. The Fab′ fragments are labeled with an activated oligonucleotideas produced according to Example 3. This single-stranded DNA (=ssDNA)bears a thiol-reactive maleimido group that reacts with the cysteines ofthe Fab′ hinge region. In order to obtain high percentages ofsingle-labeled Fab′ fragments the relative molar ratio of ssDNA toFab′-fragment is kept low. Purification of single-labeled Fab′ fragments(ssDNA: Fab′=1:1) occurs via ion exchange chromatography (column: Source15 Q PE 4.6/100, Pharmacia/GE). Verification of efficient purificationis achieved by analytical gel filtration and SDS-PAGE.

B) Assembly of an Anti-pIGF-1R Dual Binder. The anti-pIGF-1R dual binderis based on two Fab′ fragments that target different epitopes of theintracellular domain of IGF-1R: Fab′ 8.1.2 detects a phosphorylationsite (pTyr 1346) and Fab′ 1.4.168 a non-phospho site of the said targetprotein. The Fab′ fragments have been covalently linked tosingle-stranded DNA (ssDNA): Fab′ 1.4.168 to a 17mer ssDNA comprisingSEQ ID NO:6 and containing fluorescein as an fluorescent marker and Fab′8.1.2 to a 19mer ssDNA comprising SEQ ID NO:5 and containing Cy5 asfluorescent marker. In the following, these Fab's with covalently bound17mer or 19mer ssDNA are named ssFab′ 1.4.168 and ssFab′ 8.1.2respectively. Dual binder assembly is mediated by a linker (i.e. abridging construct comprising two complementary ssDNA oligonucleotides(SEQ ID NOs: 7 and 8, respectively) that hybridize to the correspondingssDNAs of the ssFab′ fragments. The distance between the two ssFab′fragments of the dual binder can be modified by using spacers, e.g.C18-spacer or DNAs of different length, respectively.

For assembly evaluation the dual binder components ssFab′ 8.1.2, ssFab′1.4.168 and the linker constructs (I) (=linker 17 of example 2.4) 5′-GCAG AAG CAT TAA TAG ACT T(-Bi)-TGG ACG ACG ATA GAA CT-3′ and (II)(=linker 10 of example 2.4) 5′-G CAG AAG CAT TAA TAGACT-(T20)-T(-Bi)-(T20)-TGG ACG ACG ATA GAA CT-3′ were mixed in equimolarquantities at room temperature. After a 1 minute incubation step thereaction mix was analyzed on an analytical gel filtration column(Superdex™ 200, 10/300 GL, GE Healthcare). Comparison of the elutionvolumes (V_(E)) of the single dual binder components with the V_(E) ofthe reaction mix demonstrates that the dual binder has been formedsuccessfully (FIG. 1). (The biotinylated thymidine (T-(Bi)) in themiddle of both of the linkers is without function in these experiments.)

2.6 Biacore™ Experiment Assessing Binding of Anti-pIGF-1R Dual Binder toImmobilized IGF-1R and IR Peptides

For this experiment a Biacore™ 2000 instrument (GE Healthcare) was usedwith a Biacore™ SA sensor mounted into the system at T=25° C.Preconditioning occurred at 100 μl/min with 3×1 min injection of 1 MNaCl in 50 mM NaOH and 1 min 10 mM HCl.

HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween® 20 wasused as system buffer. The sample buffer was identical with the systembuffer. The Biacore™ 2000 System was driven under the control softwareV1.1.1.

Subsequently biotinylated peptides were captured on the SA surface inthe respective flow cells. 16 RU of IGF-1R(1340-1366)[1346-pTyr;Glu(Bi-PEG-1340]amid (i.e. the -1346 tyrosine phosphorylated-peptide ofSEQ ID NO:11 comprising a PEG-linker bound via glutamic acidcorresponding to position 1340 and being biotinylated at the other endof the linker) was captured on flow cell 2. 18 RU of IGF-1R(1340-1366);Glu(Bi-PEG-1340]amid (i.e. the -1346 tyrosine non-phosphorylated-peptideof SEQ ID NO:11 comprising a PEG-linker bound via glutamic acidcorresponding to position 1340 and being biotinylated at the other endof the linker) was captured on flow cell 3. RU ofhIR(1355-1382)[1361-pTyr; Glu(Bi-PEG-1355]amid (i.e. the -1361 tyrosinephosphorylated-peptide of SEQ ID NO:12 comprising a PEG-linker bound viaglutamic acid corresponding to position 1355 of human insulin receptorand being biotinylated at the other end of the linker) was captured onflow cell 4. Finally all flow cells were saturated with d-biotin.

For the Dual Binder formation the assembly protocol as described inExample 2.5 was used. When individual runs with only one of the twossFab's were performed, the absence or presence of linker DNA did notaffect the association or dissociation curves (data not shown).

100 nM of analyte (i.e. in these experiments a bi-valent dual bindingagent) in solution was injected at 50 μl/min for 240 sec associationtime and dissociation was monitored for 500 sec. Efficient regenerationwas achieved by using a 1 min injection step at 50 μl/min with 80 mMNaOH. Flow cell 1 served as a reference. A blank buffer injection wasused instead of an antigen injection to double reference the data bybuffer signal subtraction.

In each measurement cycle one of the following analytes in solution wasinjected over all 4 flow cells: 100 nM ssFab′ 8.1.2, 100 nM ssFab′1.4.168, a mixture of 100 nM ssFab′ 8.1.2 and 100 nM ssFab′, 100 nMbi-valent binding agent consisting of ssFab′ 8.1.2 and ssFab′ 1.4.168hybridized on linker (III) (5′-G CAG AAG CAT TAA TAGACT-T(20)-T(-Dig)-(T20)-TGG ACG ACG ATA GAA CT-3′(=linker 15 of example2.4)), and 100 nM bi-valent binding agent consisting of ssFab′ 8.1.2 andssFab′ 1.4.168 hybridized on linker (IV) (5′-G CAG AAG CAT TAA TAGACT-T(-Dig)-TGG ACG ACG ATA GAA CT-3′(=linker 16 of example 2.4)),respectively. (The digoxigenylation of the middle thymidine (T(-Dig)) inthe above linkers is without relevance to these experiments.)

The signals were monitored as time-dependent BIAcore™ sensorgrams.

Report points were set at the end of the analyte association phase(Binding Late, BL) and at the end of the analyte dissociation phase(Stability Late, SL) to monitor the response unit signal heights of eachinteraction. The dissociation rates kd (1/s) were calculated accordingto a linear 1:1 Langmuir fit using the Biacore™ evaluation software 4.1.The complex halftimes in minutes were calculated upon the formulaln(2)/(60*kd).

The sensorgrams (FIG. 2-5) show a gain in both specificity and complexstability in pIGF-1R binding when ssFab′ 1.4.168 and ssFab′ 1.4.168 areused in form of a dual binder (=bi-valent binding agent), probably dueto the underlying cooperative binding effect. Fab′ 1.4.168 alone showsno cross reactivity for the pIR peptide but does not discriminatebetween the phosphorylated and unphosphorylated form of IGF-1R (T1/2dis=3 min in both cases). Fab′ 8.1.2, however, binds only to thephosphorylated version of the IGF1-R peptide but exhibits some undesiredcross reactivity with phosphorylated Insulin Receptor. The Dual Binderdiscriminates well between the pIGF-1R peptide and both other peptides(see FIG. 4) and thus helps to overcome issues of unspecific binding.Note that the gain in specificity is lost when both Fab's are appliedwithout linker DNA (FIG. 5). The gain in affinity of the Dual Bindertowards the pIGF-1R peptide manifests in increased dissociation halftimes compared to individual Fab's and the Fab′ mix omitting the linkerDNA (FIG. 3 and FIG. 5). Although the tested Dual Binders with twodifferent DNA linker lengths share an overall positive effect on targetbinding specificity and affinity, the longer linker ((III) withT40-T-Dig as a spacer) (i.e. linker 15 of example 2.4) seems to beadvantageous with respect to both criteria.

2.7 Biacore™ Assay Sandwich of M-1.4.168-IgG and M-8.1.2-IgG

A Biacore™ T100 instrument (GE Healthcare) was used with a Biacore™ CM5sensor mounted into the system. The sensor was preconditioned by a 1 mininjection at 100 μl/min of 0.1% SDS, 50 mM NaOH, 10 mM HCl and 100 mMH3PO4.

The system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mMEDTA, 0.05% Tween® 20). The sample buffer was the system buffer.

The Biacore™ T100 System was driven under the control software V1.1.1.Polyclonal rabbit IgG antibody <IgGFCγM>R (Jackson ImmunoResearchLaboratories Inc.) at 30 pg/ml in 10 mM Na-Acetate pH 4.5 wasimmobilized at 10 000 RU on the flow cells 1, 2, 3, and 4, respectively,via EDC/NHS chemistry according to the manufacturer's instructions.Finally, the sensor surface was blocked with 1M ethanolamine. Thecomplete experiment was driven at 13° C.

500 nM primary mAb M-1.004.168-IgG was captured for 1 min at 10 μl/minon the <IgGFCγM>R surface. 3 μM of an IgG fragment mixture (of IgGclasses IgG1, IgG2a, IgG2b, IgG3) containing blocking solution wasinjected at 30 μl/min for 5 min. The peptideIGF-1R(1340-1366)[1346-pTyr; Glu(Bi-PEG-1340]amid was injected at 300 nMfor 3 min at 30 μl/min. 300 nM secondary antibody M-8.1.2-IgG wasinjected at 30 μl min. The sensor was regenerated using 10 mMGlycine-HCl pH 1.7 at 50 μl/min for 3 min.

FIG. 6 describes the assay setup. In FIG. 7. the measurement results aregiven. The measurements clearly indicate, that both monoclonalantibodies are able to simultaneously bind two distinct, unrelatedepitopes on their respective target peptide. This is a prerequisite toany latter experiments with the goal to generate cooperative bindingevents.

2.8 Biacore™ Assay Dual Binder on Sensor Surface

A Biacore™ 3000 instrument (GE Healthcare) was used with a Biacore™ SAsensor mounted into the system at T=25° C. The system was preconditionedat 100 μl/min with 3×1 min injection of 1 M NaCl in 50 mM NaOH and 1 min10 mM HCl.

The system buffer was HBS-ET (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mMEDTA, 0.05% Tween® 20). The sample buffer was the system buffer.

The Biacore™ 3000 System was driven under the control software V4.1.

124 RU amino-PEO-biotin were captured on the reference flow cell 1. 1595RU biotinylated 14.6 kDa T0-Bi 37-mer ssDNA-Linker (I) (5′-G CAG AAG CATTAA TAG ACT-T(-Bi)-TGG ACG ACG ATA GAA CT-3′) (=linker 17 of example2.4) and 1042 RU biotinylated 23.7 kDa T40-Bi 77-mer ssDNA-Linker (II)(5′-G CAG AAG CAT TAA TAG ACT-T(20)-(Biotin-dT)-(T20)-TGG ACG ACG ATAGAA CT-3′=linker 10 of example 2.4) were captured on different flowcells.

300 nM ssFab′ 8.1.2 and 300 nM ssFab′ 1.004.168 were injected into thesystem at 50 μl/min for 3 min. As a control only 300 nM ssFab′ 8.1.2 or300 nM ssFab′ 1.004.168 was injected to test the kinetic contribution ofeach ssFab. As a control, buffer was injected instead of the ssFabs. Thepeptides pIR(1355-1382)[1361-pTyr]amid and IGF-1R(1340-1366)amid,respectively, were injected into system at 50 μl/min for 4 min, free insolution, in concentration steps of 0 nM, 4 nM, 11 nM, 33 nM (twice),100 nM and 300 nM. In another set of experiments to measure theaffinities versus the peptide pIGF-1R(1340-1366)[1346-pTyr]amid theconcentration steps of 0 nM, 0.4 nM, 1.1 nM, 3.3 nM (twice), 10 nM and30 nM were used.

The dissociation was monitored at 50 μl/min for 5.3 min. The system wasregenerated after each concentration step with a 12 sec pulse of 250 mMNaOH and was reloaded with ssFab′ ligand.

FIG. 8 schematically describes the assay setup on the Biacore™instrument. The table given in FIG. 9 shows the quantification resultsfrom this approach. FIGS. 10, 11 and 12 depict exemplary Biacore™results from this assay setup using the T40 dual binding agent.

The table in FIG. 9 demonstrates the benefits of the dual binderconcept. The T40 dual binding agent (a dual binding agent with linker 10of example 2.4, i.e. a linker with a spacer of T20-Biotin-dT-T20)results in a 2-fold improved antigen complex halftime (414 min) and a3-fold improved affinity (10 μM) as compared to the TO dual bindingagent (i.e. a dual binding agent with linker 16 of example 2.4) with 192min and 30 μM, respectively. This underlines the necessity to optimizethe linker length to generate the optimal cooperative binding effect.

The T40 dual binding agent (i.e. the dual binding agent comprising theT40-Bi linker (linker 10 of example 2.4)) exhibits a 10 μM affinityversus the phosphorylated IGF-1R peptide (table in FIG. 9, FIG. 10).This is a 2400-fold affinity improvement versus the phosphorylatedinsulin receptor peptide (24 nM) and a 100-fold improvement versus thenon-phosphorylated IGF-1R peptide.

Therefore, the goal to increase specificity and affinity by thecombination of two distinct and separated binding events is achieved.

The cooperative binding effect especially becomes obvious from thedissociation rates against the phosphorylated IGF-1R peptide, where thedual binder shows 414 min antigen complex halftime, versus 0.5 min withthe monovalent binder 8.1.2 alone and versus 3 min with the monovalentbinder 1.4.168 alone, respectively.

Furthermore, the fully assembled construct roughly multiplies itsdissociation rates kd (1/s), when compared to the singly Fab′ hybridizedconstructs (FIGS. 10, 11, 12 and table in FIG. 9). Interestingly, alsothe association rate ka (1/Ms) slightly increases when compared to thesingle Fab′ interaction events, this may be due to an increase of theconstruct's molecular flexibility.

A diagnostic system using an intense washing procedure should definitelyfoster the high performance of the T40 dual binding agent, in contrastto individual (monovalent) Fab′ molecules. The hybridized construct,i.e. a bi-valent binding agent according to the present disclosure,generates a specific and quite stable binding event, while themonovalent binders more rapidly dissociate, e.g. they are more rapidlywashed away.

Example 3 Bi-Valent Binding Agent to HER2

3.1 Assembly of an Anti-HER2 Bi-Valent Binding Agent

Two monoclonal antibodies binding to human HER2 (ErbB2 or p185^(neu)) atdifferent, non-overlapping epitopes A and B were used. The firstantibody is anti-HER2 antibody 4D5 (huMAb4D5-8, rhuMAb HER2, trastuzumabor HERCEPTIN®; see U.S. Pat. No. 5,821,337 incorporated herein byreference in its entirety).

The “4D5 epitope” is the region in the extracellular domain of ErbB2 towhich the anti-HER2 antibody 4D5 (ATCC CRL 10463) binds. This epitope isclose to the transmembrane domain of ErbB2.

The second antibody is anti-HER2 antibody 2C4 (pertuzumab®). Theantibody 2C4 and in particular the humanized variants thereof aredescribed in detail in WO 01/00245 incorporated herein by reference inits entirety. 2C4 is produced by the hybridoma cell line deposited withthe American Type Culture Collection, Manassass, Va., USA under ATCCHB-12697. Examples of humanized 2C4 antibodies are provided in Example 3of WO 01/00245 (incorporated herein by reference in its entirety). Thehumanized anti-HER2 antibody 2C4 is also called Pertuzumab.

Pertuzumab (formerly 2C4) is the first of a new class of agents known asHER dimerization inhibitors (HDIs). Pertuzumab binds to HER2 at itsdimerization domain, thereby inhibiting its ability to form active dimerreceptor complexes and thus blocking the downstream signal cascade thatultimately results in cell growth and division (see Franklin, M. C.,Cancer Cell 5 (2004) 317-328). Pertuzumab is a fully humanizedrecombinant monoclonal antibody directed against the extracellulardomain of HER2.

Purification of the monoclonal antibodies from culture supernatant canbe carried out using state of the art methods of protein chemistry.

The purified monoclonal antibodies are protease digested with eitherpre-activated papain or pepsin yielding F(ab′)₂ fragments. These aresubsequently reduced to Fab′-fragments with a low concentration ofcysteamin at 37° C. The reaction is stopped by separating the cysteaminon a Sephadex G-25 column (GE Healthcare) from thepolypeptide-containing part of the sample.

The obtained Fab′-fragments are conjugated with the activated ssDNApolynucleotides.

a) Anti-HER2 Antibody 4D5 Fab′-ssDNA-Conjugate

For preparation of the anti-HER2 antibody 4D5 Fab′-ssDNA-conjugate aderivative of SED ID NO:5 is used, i.e. 5′-AGT CTA TTA ATG CTT CTGC(=SEQ ID NO:5)-XXX-Y-Z-3′, wherein X=propylene-phosphate introduced viaphosphoramidite C3(3-(4,4′-dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research), wherein Y=5′-amino-modifier C6 introduced via(6-(4-monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(Glen Research), and whereinZ=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced viaSulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(ThermoFischer).

b) Anti-HER2 Antibody 2C4 Fab′-ssDNA-Conjugate

For the preparation of the anti-HER2 antibody 2C4 Fab′-ssDNA-conjugate Ba derivative of SEQ ID NO:6 is used, i.e. 5′-Y-Z-XXX-AGT TCT ATC GTC GTCCA-3′, wherein X=propylene-phosphate introduced via Phosphoramidite C3(3-(4,4′-Dimethoxytrityloxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research), wherein Y=5′-Amino-Modifier C6 introduced via(6-(4-Monomethoxytritylamino)hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite(Glen Research), and whereinZ=4[N-maleinimidomethyl]cyclohexane-1-carboxy introduced viaSulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate(ThermoFischer).

The polynucleotides of SEQ ID NO:5 or SEQ ID NO:6, respectively, havebeen synthesized by state of the art polynucleotide synthesis methods.The introduction of the maleinimido group was done via reaction of theamino group of Y with the succinimidyl group of Z which was incorporatedduring the solid phase polynucleotide synthesis process.

The single-stranded DNA constructs bear a thiol-reactive maleimido groupthat reacts with a cysteine of the Fab′ hinge region generated by thecysteamine treatment. In order to obtain a high percentage ofsingle-labeled Fab′-fragments the relative molar ratio of ssDNA toFab′-fragment is kept low. Purification of single-labeled Fab′-fragments(ssDNA:Fab′=1:1) occurs via anion exchange chromatography (column:MonoQ, GE Healthcare). Verification of efficient labeling andpurification is achieved by analytical gel filtration chromatography andSDS-PAGE.

3.2 Biomolecular Interaction Analysis

For this experiment a Biacore T100 instrument (GE Healthcare) was usedwith a Biacore SA sensor mounted into the system at T=25° C.Preconditioning occurred at 100 μl/min with 3×1 min injection of 1 MNaCl in 50 mM NaOH, pH 8.0 followed by a 1 min injection of 10 mM HCl.The system buffer was HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mMEDTA, 0.05% P 20). The sample buffer was the system buffer supplementedwith 1 mg/ml CMD (Carboxymethyldextrane).

Biotinylated ss-L-DNA linkers were captured on the SA surface in therespective flow cells. Flow cell 1 was saturated with amino-PEO-Biotin(PIERCE).

40 RU of the biotinylated 37mer oligonucleotide linker (linker 3 ofexample 2.4) were captured on flow cell 2. 55 RU of the biotinylated77mer oligonucleotide linker (linker 5 of example 2.4) were captured onflow cell 3. 60 RU of biotinylated 97mer oligonucleotide linker (linker6 of example 2.4) were captured on flow cell 4.

250 nM anti-HER2 antibody 4D5-Fab′-ss-L-DNA was injected into the systemfor 3 min. 300 nM anti-HER2 antibody 2C4-Fab′-ss-L-DNA was injected intothe system at 2 μl/min for 5 min. The DNA-labeled Fab fragments wereinjected alone or in combination.

As a control only 250 nM anti-HER2 antibody 4D5-Fab′-ss-D-DNA and 300 nManti-HER2 antibody 2C4-Fab′-ss-D-DNA was injected into the system. As afurther control, buffer was injected instead of the DNA-labeled Fabfragments. After hybridization of the ss-L-DNA-labeled Fab fragments onthe respective ss-L-DNA bi-linkers, the analyte in solution hHER2-ECDwas injected at different concentration series from 24 nM, 8 nM, 3 nM, 1nM, 0.3 nM, 0 nM into the system for 3.5 min association phase at 100μl/min. The dissociation phase was monitored at 100 μl/min for 15 min.The system was regenerated by a 30 sec injection at 20 μl/min of 100 mMgycine buffer (Glycine pH 11, 150 mM NaCl), followed by a second 1 mininjection of water at 30 μl/min.

The signals were measured as analyte concentration-dependent, timeresolved sensorgrams. The data was evaluated using the BiacoreBiaevaluation software 4.1. As a fitting model a standard Langmuirbinary binding model was used.

Results:

No HER2-ECD interaction could be observed when ss-D-DNA labeled Fabfragments were injected into the system, because the ss-D-DNA-labeledFab fragments did not hybridize with spiegelmeric ss-L-DNA linkerspresented on the sensor surface.

Table 3.: Kinetic results of the dual binder experiment. Linker: Surfacepresented biotinylated ss-L-DNA polynucleotide linker,Oligo_(—)37mer-Bi, Oligo_(—)77mer-Bi and Oligo_(—)97mer-Bi differing inlinker length as described above. ss-L-DNA-Fab: 2C4-ss-L-DNA: anti-HER2antibody 2C4-Fab′-ss-L-DNA labeled with 19mer-Fluorescein. 4D5-ss-L-DNA:anti-HER2 antibody 4D5-Fab′-ss-L-DNA labeled with 17mer-Fluorescein.4D5-+2C4-ss-L-DNA relates to the surface bound dual binding agentcomprising the combination of both monovalent anti-HER2-antibodyfragments.

In Table 3 the following abbreviations are used: LRU: mass in responseunits, which is hybridized on the sensor surface. Antigen: a 87 kDaHER2-ECD was used as analyte in solution. ka: association rate in(1/Ms). kd: dissociation rate in (1/s). t1/2 diss: antigen complexhalftime calculated in hours according to the solution In(2)/kd*3600 ofa first order kinetic equation. KD: affinity in molar. KD: affinitycalculated in picomolar. Rmax: Maximum analyte response signal atsaturation in response units (RU). MR: Molar Ratio, indicating thestoichiometry of the interaction. Chi2, U-value: quality indicator ofthe measurements.

TABLE 3 k_(a) k_(d) t′₂-diss K_(D) R_(max) Chi² Linker ss-L-DNA-Fab LRUAntigen 1/Ms 1/s hours K_(D) M pM RU MR RU² Oligo_35mer-Bi 4D5- +2C4-ss-L-DNA 84 Her2-ECD 5.9E+05 6.7E−05 3 1.1E−10 100 59 0.9 0.2Oligo_35mer-Bi 4D5-ss-L-DNA 16 Her2-ECD 4.0E+05 3.4E−05 6 8.5E−11 100 291.2 0.1 Oligo_35mer-Bi 2C4-ss-L-DNA 31 Her2-ECD 3.3E+05 3.6E−05 51.1E−10 100 26 0.6 0.03 Oligo_75mer-Bi 4D5- + 2C4-ss-L-DNA 87 Her2-ECD5.1E+05 4.6E−08 4164 9.1E−14 0.1 65 1.0 0.1 Oligo_75mer-Bi 4D5-ss-L-DNA16 Her2-ECD 2.9E+05 6.1E−05 3 2.1E−10 200 31 1.3 0.04 Oligo_75mer-Bi2C4-ss-L-DNA 29 Her2-ECD 3.8E+05 6.3E−05 3 1.6E−10 200 32 0.7 0.03Oligo_95mer-Bi 4D5- + 2C4-ss-L-DNA 76 Her2-ECD 5.0E+05 4.9E−08 39429.9E−14 0.1 58 1.0 0.1 Oligo_95mer-Bi 4D5-ss-L-DNA 14 Her2-ECD 3.0E+059.5E−05 2 3.1E−10 300 28 1.3 0.03 Oligo_95mer-Bi 2C4-ss-L-DNA 28Her2-ECD 3.8E+05 6.8E−05 3 1.8E−10 300 27 0.6 0.03

In the above Table 35mer, 75mer and 95mer, respectively should read37mer, 77mer and 97mer, respectively.

The biacore data for the 37mer dual binder HER2-ECD interaction (i.e.for a binder with a linker consisting solely the hybridization sequencesmotives attached to the binders and a central biotinylated thymidin)indicate that this dual binding agent shows no improvement in kineticperformance. This is most likely due to the insufficient linker lengthand the lack in flexibility of the 37mer linker.

The biacore data for the 77mer dual binder HER2-ECD interaction (i.e.for a binder with a linker comprising twice 20 thymidines a centralbiotinylated thymidin to increase the linker length) indicate, that thisdual binding agent shows a dramatic improvement in its kineticperformance. This is most likely due to an optimal linker length and theflexibility of this 77mer linker.

The biacore data for the 97mer dual binder HER2-ECD interaction (i.e.for a binder with a linker comprising twice 30 thymidines a centralbiotinylated thymidin to increase the linker length) indicate, that thisdual binding agent shows a dramatic improvement in its kineticperformance. This is most likely due to an optimal linker length and theflexibility of this 97mer linker.

The data in Table 3 provide evidence for the presence of a cooperativebinding event. Despite the Rmax values of the fully established dualbinders are roughly double the signal height of the singly Fab-armedconstructs, the Molar Ratio values are exactly 1 (MR=1). This is a clearevidence for the presence of a simultaneous, cooperative binding eventof both Fab fragments. The dual binder counts is a single molecule witha 1:1 Langmuir binding stoichiometry. Despite having 2 independentlybinding HER2 interfaces no inter molecule binding between one dualbinder and two HER2 domains can be detected.

The avidity constants for synergizing pairs of monoclonal antibodies orfor a chemically cross-linked bispecific F(ab′)2 is generally only up to15 times greater than the affinity constants for the individualmonoclonal antibodies, which is significantly less than the theoreticalavidity expected for ideal combination between the reactants (Cheong, H.S., et al., Biochem. Biophys. Res. Commun. 173 (1990) 795-800). Withoutbeing bound by this theory one reason for this might be that theindividual epitope/paratope interactions involved in a synergisticbinding (resulting in a high avidity) must be orientated in a particularway relative to each other for optimal synergy.

Furthermore, the data presented in Table 3 provides evidence, that theshort 37mer linker, which consists just from the ss-L-DNA hybridizationmotives doesn't show enough flexibility or/and linker length to producethe cooperative binding effect. The 37mer linker is a rigid, doublehelix L-DNA construct. The hybridization generates a double L-DNA helix,which is shorter and less flexible than the ss-L-DNA sequence. The helixshows reduced degrees of freedom and can be seen as a rigid linkerconstruct. Table 3 shows, that the 37mer linker isn'table to generate acooperative binding event. The fully established 37mer dual binder showsthe same affinity like only the singly hybridized constructs.

Extending the linker length by a highly flexible poly-T ss-L-DNA to forma 77mer and a 97mer, respectively, provides for an increase in affinityand especially in antigen complex stability kd (1/s).

The chi2 values indicate a high quality of the measurements. Allmeasurements show extremely small errors. The data can be fitted to aLangmuir 1:1 fitting model residuals deviate only +/−1 RU, small chi2values and only 10 iterative calculations were necessary for obtainingthe data.

A cooperative binding effect works according to the physical law, thatthe free binding energies ΔG1 and ΔG2 summarize. The affinitiesmultiply: Kdcoop=KD1×KD2. Furthermore, the dissociation rates alsomultiply: kd coop=kd1×kd 2. This is exactly observable in the 77mer and97mer linker experiment. This results in very long complex half-lifes of4146 hours (173 days) and 3942 hours (164 days), respectively. Theaffinities are in the range of 100 fmol/l. It is obvious, that acooperative binding event occurs.

The association rates of all dual binding agents are faster, whencompared to the singly hybridized constructs. Despite showing a highermolecular weight the association rate increases.

Here we could show, that tratsuzumab and pertuzumab linked together in acomplex as reported herein simultaneously binds to the HER-2extracellular domain (ECD). Both Fab fragments bind to genuine epitopeson the HER2-ECD. Additionally both Fab fragments strongly differ intheir binding angles. By using the optimal 77mer linker (about 30 nm inlength) ss-L-DNA and its beneficial flexibility and length properties acooperative binding event could be shown.

Hence cooperative binding between Herceptin-Fab and Pertuzumab-Fablinked together via a highly flexible ss-L-DNA linker could be shown.

While this disclosure has been described as having an exemplary design,the present disclosure may be further modified within the spirit andscope of this disclosure. This application is therefore intended tocover any variations, uses, or adaptations of the disclosure using itsgeneral principles. Further, this application is intended to cover suchdepartures from the present disclosure as come within the known orcustomary practice in the art to which this disclosure pertains.

What is claimed is:
 1. A binding agent comprising Formula:A-a′:a-S-b:b′-B:X(n), wherein A is a monovalent binder; B is amonovalent binder; S is a spacer of at least 1 nm in length; (n) is aninteger and at least 1; - represents a covalent bond; a′:a is a bindingpair; b:b′ is a binding pair, wherein a′ and a do not interfere with thebinding of b to b′ and b′ and b do not interfere with the binding of a′to a; (: X) denotes a functional moiety bound either covalently or via abinding pair to at least one of a′, a, b, b′ or S; and linker a-S-b hasa length of 6 to 100 nm.
 2. The binding agent of claim 1, wherein thespacer S is 1 to 95 nm in length.
 3. The binding agent of claim 1,wherein the a′:a binding pair and the b:b′ binding pair are selectedfrom the group consisting of leucine zipper domain dimers andhybridizing nucleic acid sequences.
 4. The binding agent of claim 1,wherein the a′:a binding pair and the b:b′ binding pair are hybridizingnucleic acid sequences and wherein the different hybridizing nucleicacid sequences of the a′:a binding pair does not hybridize with the b:b′binding pair.
 5. The binding agent of claim 1, wherein the spacer S is anucleic acid.
 6. The binding agent of claim 5, wherein the a′:a bindingpair and the b:b′ binding pair are nucleic acids.
 7. The binding agentof claim 6, wherein the monovalent binders A and B are nucleic acids. 8.The binding agent of claim 1, wherein X is a functional moiety selectedfrom the group consisting of a labeling group, a binding group and aneffector group.
 9. The binding agent of claim 1, wherein the functionalmoiety X is bound to a, b, or S.
 10. The binding agent of claim 1,wherein the functional moiety X is bound to the spacer S.
 11. Thebinding agent of claim 1, wherein the functional moiety X is covalentlybound to the spacer S.
 12. The binding agent of claim 1, wherein thefunctional moiety X is bound to the spacer S via a hybridizing nucleicacid.
 13. The binding agent of claim 1, wherein the monovalent binders Aand B are polypeptides.
 14. The binding agent of claim 13, wherein themonovalent binders A and B are Fab-fragments of monoclonal antibodies.15. The binding agent of claim 1, wherein X is a functional moietyselected from the group consisting of a labeling group, a binding groupand an effector group and the monovalent binders A and B arepolypeptides.